Exploring the Promiscuity of Phenol Hydroxylase from Pseudomonas

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Exploring the promiscuity of phenol hydroxylase from Pseudomonas stutzeri OX1 for the biosynthesis of phenolic compounds Jia Wang, Xiaolin Shen, Jian Wang, Yaping Yang, Qipeng Yuan, and Yajun Yan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00067 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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ACS Synthetic Biology

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Exploring the promiscuity of phenol hydroxylase from Pseudomonas stutzeri OX1 for the

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biosynthesis of phenolic compounds

3 Jia Wanga,b,1, Xiaolin Shena,b,1, Jian Wangc, Yaping Yangc, Qipeng Yuana,b,*, Yajun Yanc,*

4 5 6

a

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University of Chemical Technology, Beijing 100029, China

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing

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b

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

Technology, Beijing 100029, China

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c

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University of Georgia, Athens, GA 30602, USA

School of Chemical, Materials and Biomedical Engineering, College of Engineering, The

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1

JW and XS contributed equally to this work

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* Corresponding authors:

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Qipeng Yuan

19

15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China

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E-mail: [email protected]; telephone: +86-10-64437610

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Yajun Yan

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146 Riverbend Research Lab South, The University of Georgia, Athens, GA 30602, USA

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E-mail: [email protected]; telephone: +1-706-542-8293

1 ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Enzyme promiscuity plays an important role in developing biosynthetic pathways for novel

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target products. Phenol hydroxylase (PH) from Pseudomonas stutzeri OX1 is capable of ortho-

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hydroxylation of phenol and cresol isomers into counterpart catechols. A small ferredoxin-like

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protein PHQ was clustered together with the ph gene cluster in the genome of P. stutzeri OX1

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and its function was not known. In this study, we found that the existence of PHQ has a

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promotion effect on the catalytic efficiency of PH. Then, we tested the substrate range of PH

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using nine different non-natural substrates. We found that PH was a promiscuous hydroxylase

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that could catalyze ortho-hydroxylation of several non-natural substrates, including catechol, 4-

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hydroxybenzoic acid and resorcinol. On this basis, linking the catechol biosynthetic pathway

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with the hydroxylation reaction catalyzed by PH enabled construction of a novel biosynthetic

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pathway for the synthesis of pyrogallol. This work not only characterized a well-performed PH

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but also provided a promising hydroxylation platform for the production of high-value phenolic

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compounds.

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Keywords: Phenol hydroxylase, Promiscuity, Pyrogallol, Phenolic compounds


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Phenolic compounds possess an aromatic ring bonded with one or more hydroxyl groups in their

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structures. Those compounds are of significant value in pharmaceutical industry owing to their

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outstanding biological properties such as antioxidant, antibacterial, anti-inflammatory, anti-aging,

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anti-cancer, anti-diabetic and anti-ulcer activities1-3. However, the extremely low abundance of

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those compounds in nature has limited their large-scale commercial applications. Although

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chemical synthesis has been widely used in production of natural products, regio- or

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enantioselective hydroxylation of aromatic compounds via chemical approaches is still a big

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challenge due to the poor selectivity4. Alternatively, development of biotransformations for the

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hydroxylation reactions under mild and environmental friendly processing conditions have

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attracted tremendous attention in recent years5-7.

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Bacterial multicomponent monooxygenases (BMMs) represent a large family of nonheme diiron

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proteins that hydroxylate a broad variety of alkanes, alkenes and aromatic compounds with high

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regio- and stereoselectivity8. Two different BMMs were identified in the genome of

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Pseudomonas stutzeri OX1, toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase

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(PH)9. Those two enzymes play important roles in aromatic hydrocarbons degradation10. The

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ToMo has been well characterized so far, which can oxidize benzene, xylene, toluene, cresol,

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2,3-dimethylphenol, 3,4-dimethylphenol, naphthalene and styrene11. In comparison, less effort

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has been made to explore the substrate spectrum of PH. The nucleotide sequence of the locus

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coding for PH has been revealed as a cluster of six open reading frames, named ph K, L, M, N, O

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and P, which are designated as six polypeptides12. The hydroxylase component is constituted by

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three polypeptides (L, N and O) that form a dimeric subcomplex (LNO)2. PH(LNO)2 alone

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without any other components is capable of converting phenol to catechol9. The polypeptide PHP



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is the NAD(P)H-oxidoreductase component in the PH complex responsible for transferring

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electrons into diiron cluster in the active site via a flavin cofactor and [2Fe-2S] center. PHP alone

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is not able to convert phenol to catechol, while supplement of PHP to PH(LNO)2 enhanced the

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catechol titer by 2.48 folds9. PHM is a small regulatory protein that is indispensable for efficient

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catalysis. Although supplement of PHM to PH(LNO)2 is almost ineffective, combinatory

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expression of all three components, PH(LNO)2, PHP and PHM resulted in a 12-fold

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improvement in catechol titer compared with expression of PH(LNO)2 alone9. Finally, PHK is an

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auxiliary protein that might be involved in incorporation of metal iron into the enzyme active site.

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Addition of PHK to PH(LNO)2, PHP and PHM increased the enzyme activity of PH by 2-3

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folds13.

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Actually, except of phKLMNOP, there exists an additional ORF at the end of the ph gene cluster

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in the genome of P. stutzeri OX1, named phQ (Figure 1A). It has been annotated as a putative

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chloroplast-type ferredoxin with a molecular mass of about 11 KDa, but its exact role is still

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elusive. Generally, it is a common observation that in the presence of accessory proteins in

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several BMMs, such as ToMOD in toluene o-xylene monooxygenase (ToMO)14 and MMOB in

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soluble methane monooxygenase (sMMO)15. Considering the favorable effects of those

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accessory proteins for BMMs, we hypothesized that may be PHQ has a promotion effect on the

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catalytic efficiency of PH. To experimentally verify our hypothesis, we first performed in vitro

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crude extract enzyme assays to test the enzyme activity of PH in the presence or absence of PHQ.

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The gene clusters phKLMNOP and phKLMNOPQ were cloned into the high copy-number

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plasmid pZE12-luc separately. They were individually introduced into E. coli BW25113 (F’),

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generating strains JX21 and JX22, respectively. As shown in Table 1, a 39.4% improvement in


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phenol hydroxylase activity was observed for the crude extracts of JX22 harboring phQ, when

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compared with that of JX21 without phQ. In addition, the in vivo maximum conversion capacity

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of those two strains were also compared by feeding experiments. As shown in Figure 1B, strain

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JX21 expressing phKLMNOP produced 157.6 mg/L catechol at 36 h. Remarkably, JX22

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expressing phKLMNOPQ generated 533.4 mg/L catechol, representing a 3.4-fold increase

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compared with the titer in strain JX21 without phQ expression. It is worth noting that the

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promotion effect is more pronounced in feeding experiments than in the crude extracts enzyme

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assays, probably due to the favorable intracellular environment with sufficient enzyme

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regeneration and cofactor supply. Those results confirmed our hypothesis that PHQ has a

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promotion effect on the catalytic efficiency of phenol hydroxylase.

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After obtaining a well-performed PH, we began to explore its promiscuity to extend its

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application in biological catalysis. According to the previous report, PH was able to convert

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phenol and a series of phenol analogs such as cresol isomers, 2,3-dimethylphenol, 2,4-

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dimethylphenol, 2,5-dimethylphenol and 3,4-dimethylphenol to their corresponding catechols10.

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Those results indicated that PH recognizes the phenol moiety and hydroxylates the substrates at

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the ortho position. To investigate the substrate range of PH, we selected nine different

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hydroxylated aromatic compounds as the substrates (Figure 2). The in vitro crude extract enzyme

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assays were performed to test the substrate specificity of PH in the presence or absence of PHQ.

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Among the nine compounds examined in this study, PH preferred catechol, 4-HBA and

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resorcinol as the substrates, and exhibited higher activity toward all three substrates when phQ

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was expressed. This further confirmed the promoting effect of PHQ for PH. The enzyme

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activities of PHKLMNOPQ toward catechol and resorcinol were around 20-fold lower than



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towards phenol despite their higher structural similarity. While, only negligible activity was

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detected when employing 4-HBA as the substrate. Other five compounds did not serve as

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substrates for the PH (Table 1). It is worth noting that we did not observe the production of

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hydroxyhydroquinone when employing the resorcinol as the substrate, indicating that PH prefer

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hydroxylate resorcinol at C2 position.

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As we mentioned above, the PH performed better in intact cells compared with in vitro catalysis.

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Therefore, we conducted feeding experiments to explore the maximum bioconversion capacity of

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PH. In this part, only three substrates, catechol, 4-HBA and resorcinol (Figure 3A) were used

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since PH did not exhibit obvious activities toward the other substrates. We employed strain JX22

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harboring gene cluster phKLMNOPQ as the producer, the substrates were individually fed into

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the cultures at a concentration of 300 mg/L at 3 h. As shown in Figure 3B, we observed the

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hydroxylation reactions rates dramatically increased from 6 to 12h. At the end of 24 h, the

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highest pyrogallol amount reached to 184.6 mg/L when using catechol as the substrate. When

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feeding 4-HBA into the cultures, the host strain JX22 generated 35.4 mg/L 3,4-DHBA. When

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employing resorcinol as the substrate, the titer of pyrogallol reached the maximum at 107.1 mg/L.

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Those results suggested that PH is a promiscuous hydroxylase that can catalyze ortho-

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hydroxylation of several hydroxylated aromatic compounds. The presence of other types of

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group in the aromatic ring dramatically decreased the enzyme activity of PH. Despite relatively

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low catalytic efficiency towards the non-native substrates catechol, 4-HBA and resorcinol, PH

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was demonstrated to accept those three molecules as the substrates. The promiscuity of PH

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discovered in this study can be applied for construction of non-natural biosynthetic pathways to

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produce several value-added chemicals.


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To examine if PH can be employed to establish de novo biosynthetic pathways, we set up the

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pyrogallol biosynthesis as a demonstration. Pyrogallol is a simple phenolic compound with

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significant chemical and pharmaceutical applications, owing to its attractive properties such as

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antioxidant, antiseptic, antibacterial, anticancer activities and oxygen absorbance16,

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recently, we achieved de novo biosynthesis of pyrogallol from simple carbon sources by

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identification of an efficient 2,3-DHBA monooxygenase. Further optimization of metabolic

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pathway enabled production of 1.04 g/L pyrogallol in shake flask experiments17. In the present

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study, we proposed an alternative and novel route to synthesize pyrogallol by linking the

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catechol biosynthetic pathway with the hydroxylation reaction catalyzed by PH (Figure 4A).

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Thus, redirection of the carbon flux from the endogenous shikimate pathway towards catechol

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biosynthesis is the first step. In our previous study, we have reported that catechol can be

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generated from salicylic acid catalyzed by an efficient salicylic acid 1-monooxygenase (encoded

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by nahG) from P. putida DOT-T118. Additionally, we found that entC (encoding isochorismate

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synthase) from E. coli and pchB (encoding isochorismate pyruvate lyase) from P. fluorescens

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were the most efficient enzymes for conversion of chorismate, a key intermediate in the

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shikimate pathway, to salicylic acid. Over-expression of entC and pchB in wildtype E. coli strain

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resulted in 158.5 mg/L salicylic acid from glycerol4. Based on those results, we speculated that

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pyrogallol can be synthesized by introduction of salicylic 1-monooxygenase and PH into the

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salicylic acid producing strain. To verify our hypothesis, shake flask experiments were

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conducted. The genes entC, pchB and codon optimized nahGopt were cloned into the medium

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copy-number plasmid pCS27, yielding plasmid pCS-EPN. The plasmids pCS-EPN and pZE-

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phklmnopq were co-transferred into E. coli BW25113 (F’) to generate strain JX23. As shown in


17

. Very


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Figure 4B, strain JX23 produced the highest pyrogallol amount at 76.7 mg/L by the end of 36 h.

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This titer was slightly decreased to 63.3 mg/L at 48 h due to the autoxidation of pyrogallol. The

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producing strain reached to a maximum cell density (OD600 = 11.7) after 24 h cultivation, during

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the next 24 h, the OD600 value was slightly declined to 9.7. In addition, we also observed that

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113.0 mg/L catechol was left in the cultures at the end of 48 h, suggesting that the catalytic

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efficiency of the PH towards catechol is the rate-limiting step in the pyrogallol biosynthetic

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pathway.

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Accessory protein required for assembly of metalloproteins but not present in the final complex

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have been reported in many proteins, such as BMMs, nitrogenase, urease and Fe/S proteins19, 20.

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In BMMs family, the exact functions of those accessory proteins were not clear though they were

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supposed to be involved in assembly of metal cofactor into the active site13. In this study, we

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found that a small protein PHQ was clustered together with the ph gene cluster in the genome of

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P. stutzeri OX1. We experimentally demonstrated that the presence of PHQ improved the

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catalytic efficiency of PH by 3.4 folds compared with the same strain carrying ph gene cluster

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devoid of the gene phQ. Our results revealed that PHQ was essential to PH for efficient catalysis.

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In the future, further studies need to be performed to elucidate the exact mechanistic function of

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PHQ.

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The enzyme catalytic promiscuity has been demonstrated as a powerful tool for development of

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novel biosynthetic routes of non-natural products21-25. In this study, we explored the substrate

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spectrum of the PH and found that it can catalyze ortho-hydroxylation of several hydroxylated

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aromatic compounds with simple structures, such as catechol, 4-HBA and resorcinol. Based on

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those results, we employed PH to construct a non-natural and novel biosynthetic pathway to 8 ACS Paragon Plus Environment

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produce pyrogallol. Combination of the catechol biosynthetic pathway with the hydroxylation

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reaction catalyzed by PH successfully achieved pyrogallol de novo biosynthesis. Although the

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pyrogallol titer obtained in this pathway is much lower than our previous report17 due to the low

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enzyme activity of PH towards catechol, the previous study has demonstrated that using the same

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pathway could produce up to 1.5 g/L muconic acid in shake flask experiments18, suggesting the

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high-level carbon flux through this pathway can be achieved if the pathway enzymes are efficient

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enough. Since the crystal structure of PH has been resolved26, we plan to apply rational protein

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engineering approach to improve its substrate specificity towards catechol in our further work.

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Additionally, we believe that PH can be further applied to construct another new pyrogallol

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biosynthetic pathway by recruiting resorcinol as the precursor. In addition, PH catalyzed

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hydroxylation of 4-HBA to 3,4-DHBA can be used for establishment of novel gallic acid27 and

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vanillyl alcohol28 biosynthetic pathways. Overall, our study constructed a promising bio-catalytic

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hydroxylation platform for bio-production of high-value phenolic compounds.

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Methods

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Media, Strains and Plasmids

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Luria-Bertani (LB) medium consists 5 g/L yeast extract, 10 g/L NaCl and 10 g/L tryptone was

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used for strain inoculation and plasmid construction. The modified M9 medium consists 5 g/L

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yeast extract, 20 g/L glycerol, 2.5 g/L glucose, 6 g/L Na2HPO4, 0.5 g/L NaCl, 3 g/L KH2PO4, 1

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g/L NH4Cl, 246.5 mg/L MgSO4·7H2O and 14.7 mg/L CaCl2·2H2O was used for feeding

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experiments and de novo biosynthesis of pyrogallol. When needed, the antibiotics ampicillin and

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kanamycin were added into the medium to the final concentration of 100 and 50 µg/mL,

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respectively. E. coli strain XL1-Blue was used for plasmid construction and E. coli BW25113

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(F’) was used as the host for feeding experiments and de novo biosynthesis of pyrogallol. High-

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copy number plasmid pZE12-luc and medium-copy number plasmid pCS27 were used for

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pathway construction. All of used strains and plasmids in this study were listed in Table 2.

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DNA manipulations

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The gene clusters phKLMNOP and phKLMNOPQ were amplified from the genome of strain

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Pseudomonas stutzeri OX1. They digested by BsiWI and XbaI was separately cloned into the

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backbone of pZE12-luc digested by Acc65I and XbaI to create plasmid pZE-phklmnop and pZE-

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phklmnopq. Plasmids pCS-EP containing entC from E. coli and pchB from P. fluorescens Migula

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and pCS-NahGopt containing nahGopt were constructed in our previous studies4, 18. The gene

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fragments entC-pchB and nahGopt were amplified from pCS-EP and pCS-NahGopt, respectively.

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Those two fragments were inserted into plasmid pCS27 using Acc65I, BamHI and HindIII to

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create plasmid pCS-EPN.

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In vitro Crude extract enzyme assays

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The plasmids pZE-phklmnop and pZE-phklmnopq were transferred into E. coli BW25113 (F’),

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separately. The fresh transformants were inoculated at 37 oC in 3 mL LB liquid medium

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containing 100 µg/mL ampicillin. Then, 200 µL of overnight cultures were transferred into 50

220

mL fresh LB medium containing 100 µg/mL ampicillin. When the optical density at 600nm

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(OD600) of cell culture reached around 0.6, 0.5 mM isopropyl β-D-1-thiogalactopyranoside

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(IPTG) was supplemented into the cultures, followed by incubation at 30 oC for overnight. Cells

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were harvested then lysed by bead beater to obtain the cells crude extracts. Protein

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concentrations were estimated by the BCA kit (Pierce Chemicals). To evaluate the in vitro

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activities of PHs towards phenol, crude extract of PHKLMNOP (1g/L) and PHKLMNOPQ (1g/L)

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was separately added into the reaction system which contains 0.5 mM phenol and 1 mM NADH,

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the final volume was adjusted to 1 ml with Tris-HCl buffer (100 mM, pH=7.0). To evaluate the

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in vitro activities of PHs towards catechol, 4-HBA and resorcinol, the concentration of crude

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extract of PH was set at 10 g/L for 4HBA, 2 g/L for catechol and resorcinol. The reaction system

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was kept at 30 °C, the reaction time was 30 min. In those conditions, the conversion ratio was

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controlled below 20% and the reaction rate was regarded as initial reaction rate. The reaction

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rates were calculated by measuring the consumption of phenol via HPLC.

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Feeding experiments

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The plasmids pZE-phklmnop or pZE-phklmnopq was transferred into E. coli strain BW25113

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(F’). The fresh colonies were inoculated into 3 ml LB liquid medium and grown at 37ºC. 200 µL

237

of overnight cultures were transferred into 20 mL modified M9 liquid medium. When OD600

238

reached 0.6, the cultures were added with 0.5 mM IPTG and transferred to a 30ºC shaker. The



239

cultures were added with the corresponding substrates at 300 mg/L every 3 h. Samples were

240

taken every few hours and the concentrations of products were analyzed by HPLC.

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De novo biosynthesis of pyrogallol

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Overnight cultures of the producing strains were incubated in 3 ml LB medium (37ºC, 270 rpm).

244

200 µL of the pre-inoculum together with appropriate antibiotics were supplemented into flasks

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containing the modified M9 medium and grown for 3 hours (37ºC, 270 rpm). IPTG was added to

246

the cultures at a final concentration of 0.5 mM at 3 h, and the cultures were transferred to a 30 ºC

247

shaker (270 rpm) till 48 hours. Samples were taken every 12 hours. OD600 values were measured

248

and the concentrations of products and intermediates were analyzed by HPLC.

249 250

Analytical procedures

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To measure the optical density at 600 nm, Genesys 10S UV-Vis Spectrophotometer (Thermo

252

Scientific, Waltham, MA) was used. The samples were centrifuged at 13,000 rpm for 20 min, the

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supernatant was analyzed and quantified by HPLC (Dionex Ultimate 3000) equipped with a

254

reverse phase ZORBAX SB-C18 column and an Ultimate 3000 Photodiode Array Detector. The

255

column temperature was set to 28 °C. Solvent A was water with 0.1% trifluoroacetic acid, and

256

solvent B was methanol. The following gradient was used at a flow rate of 1 mL/min: 5–50%

257

solvent B for 15 min, 50–5% solvent B for 1 min, and 5% solvent B for an additional 4 min. The

258

products and intermediates were quantified based on the peak areas at UV absorbance at 220 nm.


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AUTHOR INFORMATION

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Corresponding Authors

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*E-mails: [email protected] (Q. Yuan), [email protected] (Y. Yan).

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Author Contributions

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JiaW and XS conceived the study and wrote the manuscript. JiaW and XS performed the

264

experiments. JianW and YYang participated in the research. QY and YYan directed the research.

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JiaW, XS, JianW, QY and YYan revised the manuscript.

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Notes

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The authors declare no competing financial interest.

268 269

ACKNOWLEDGMENTS

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This work was supported by National Natural Science Foundation of China (21636001,

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21776008), the Program of Introducing Talents of Discipline to Universities (“111” project,

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B13005), the Program for Changjiang Scholars and Innovative Research Team in Universities in

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China (No. IRT13045). We would also like to thank the College of Engineering, The University

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of Georgia, Athens.



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Table 1. In vitro crude extract enzyme assays of PHKLMNOP and PHKLMNOPQ toward

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different substrates. Activity (µmol/min/mg protein) Substrate

277 278

Product

PHKLMNOP Phenol Catechol 31.47 ± 1.89 Catechol Pyrogallol 1.383 ± 0.248 Salicylic acid N.D. N.D. 3-HBA N.D. N.D. 4-HBA 3,4-DHBA 0.008 ± 0.001 3,4-DHBA N.D. N.D. Tyrosine N.D. N.D. p-Coumaric acid N.D. N.D. Hydroquinone N.D. N.D. Resorcinol Pyrogallol 0.872 ± 0.059 Data are presented as mean ± s.d. (n = 3). N.D. = Not detected.


PHKLMNOPQ 43.87 ± 5.56 2.232 ± 0.181 N.D. N.D. 0.015 ± 0.003 N.D. N.D. N.D. N.D. 0.936 ± 0.580

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279 280


Table 2. List of strains and plasmids used in this study. Strain

Genotype

Reference

BW25113 (F’)

rrnBT14 ∆lacZWJ16 hsdR514 ∆araBADAH33 ∆rhaBADLD78 F‫ ׳‬Yale CGSC [traD36 proAB lacIqZ∆M15 Tn10(Tetr)]

XL1-Blue

recA1 endA1gyrA96thi-1hsdR17supE44relA1lac

Stratagene

[F’ proAB lacIqZDM15Tn10 (TetR)] JX21

BW25113 (F’) harboring pZE-phklmnop

This study

JX22

BW25113 (F’) harboring pZE-phklmnopq

This study

JX23

BW25113 (F’) harboring pCS-EPN and pZE-phklmnopq

This study

Plasmid

Description

Reference

pZE12- luc

pLlacO-1; luc; ColE1 ori; AmpR

2

pCS27

pLlacO-1; p15A ori; KanR

21

pCS-EP

pCS27 carrying entC from E. coli and pchB from P. fluorescens

4

Migula pCS-NahGopt

pCS27 carrying nahGopt from P. putida DOT-T1E

18

pZE-phklmnop

pZE12-luc carrying phklmnop from P. stutzeri OX1

This study

pZE-phklmnopq

pZE12-luc carrying phklmnopq from P. stutzeri OX1

This study

pCS-EPN

pCS27 carrying entC from E. coli, pchB from P. fluorescens

This study

Migula and nahGopt from P. putida DOT-T1E 281



282

Figure Legends

283 284

Figure 1. The promotion effect of PHQ on phenol hydroxylase (PH) activity. (A) The PH operon

285

in the genome of Pseudomonas stutzeri OX1. (B) Production curves of catechol via feeding

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experiments by employment of PHKLMNOP and PHKLMNOPQ. The data were generated from

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three independent experiments (n=3; s.d. represented by ±).

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Figure 2. The native and possible substrates of PH.

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Figure 3. Exploring the promiscuity of PH. (A) The verified substrates and reactions catalyzed

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by PH in this study. The asterisk mark indicates the hydroxylation position. (B) The results of

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feeding experiments for PH catalyzed ortho-hydroxylation of catechol, 4-HBA, and resorcinol.

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The data were generated from three independent experiments (n=3; s.d. represented by ±).

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Figure 4. Employment of PH to construct a novel biosynthetic pathway for the production of

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pyrogallol. (A) The proposed pyrogallol biosynthetic pathway. (B) The results of shake flask

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experiments for de novo biosynthesis of pyrogallol. PPP pathway: pentose phosphate pathway;

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E4P: erythrose-4-phosphate; PEP: phosphoenolpyruvate; DAHP: 3-deoxy-D-heptulosonate-7-

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phosphate, EntC: isochorismate synthase, PchB: isochorismate pyruvate lyase. NahG: salicylate

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1-monooxygenase, PH: phenol hydroxylase. The blue arrow indicated heterologous pathways.

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The data were generated from three independent experiments (n=3; s.d. represented by ±).


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Exploring the Promiscuity of Phenol Hydroxylase from Pseudomonas

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