Biotech in China 2021, at the beginning of the 14th five-year period ("145").

As China assumes a more and more dominant role in global science, this mini-review attempts to provide a bird's eye view on how the bio-digital revolution impacts China's biosciences and bioindustry. Triggered by top-down political programs and the buildup of an impressive infrastructure in science, information technology, and education, China's biomedical and MedTech industries prosper. Plant and animal breeding programs transform agriculture and food supply as much as the Internet of things, and synthetic biology offers new opportunities for the manufacturing of specialty chemicals within the Chinese version of a "bioeconomy." It is already becoming apparent that the new five-year period "145" (2021-2025) will further emphasize emission control, bioenvironmental protection, and more supply of biomass-derived energy. This review identifies key drivers in China's government, industry, and academia behind these developments and details many access points for deeper studies. KEY POINTS: Biotechnology in China Biomedical technology New five-year period.

Isolation of an organic solvent-tolerant lipolytic enzyme from uncultivated microorganism.

Although the use of lipases as biocatalysts has frequently been proposed, it is yet scarcely being implemented in industrial processes. This is mainly due to the difficulties associated with the discovery and engineering of new enzymes and the lack of versatile screening methods. In this study, we screened the available strategy from a metagenomic pool for the organic solvent-tolerant lipase with enhanced performance for industrial processes. A novel lipase was identified through functional screening from a metagenomic library of activated sludge in an Escherichia coli system. The gene encoding the lipase from the metagenomic pool, metalip1, was sequenced and cloned by PCR. Metalip1 encoding a polypeptide of 316 amino acids had typical residues essential for lipase such as pentapeptide (GXSXGG) and catalytic triad sequences (Ser160, Asp260, and His291). The deduced amino acid sequence of metalip1 showed high similarity to a putative lipase from Pseudomonas sp. CL-61 (80 %, ABC25547). Metalip1 was expressed in E. coli BL21 (DE3) with a his-tag and purified using a Ni-NTA chelating column and characterized. This enzyme showed high expression level and solubility in the heterologous E. coli host. This enzyme was active over broad organic solvents. Among organic solvents examined, dimethyl formamide was the best organic solvent for metalip1. We showed that function-based strategy is an effective method for fishing out an organic solvent-tolerant lipase from the metagenomic library. Also, it revealed high catalytic turnover rates, which make them a very interesting candidate for industrial application.

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system.

12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7ß-hydroxysteroid dehydrogenase (7ß-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.

Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane.

Oxygenases-based Escherichia coli whole-cell biocatalyst can be applied for catalysis of various commercially interesting reactions that are difficult to achieve with traditional chemical catalysts. However, substrates and products of interest are often toxic to E. coli, causing a disruption of cell membrane. Therefore, organic solvent-tolerant bacteria became an important tool for heterologous expression of such oxygenases. In this study, the organic solvent-tolerant Bacillus subtilis 3C5N was developed as a whole-cell biocatalyst for epoxidation of a toxic terminal alkene, 1-hexene. Comparing to other hosts tested, high level of tolerance towards 1-hexene and a moderately hydrophobic cell surface of B. subtilis 3C5N were suggested to contribute to its higher 1,2-epoxyhexane production. A systematic optimization of reaction conditions such as biocatalyst and substrate concentration resulted in a 3.3-fold increase in the specific rate. Co-expression of glucose dehydrogenase could partly restored NADPH-regenerating ability of the biocatalyst (up to 38 % of the wild type), resulting in approximately 53 % increase in specific rate representing approximately 22-fold increase in product concentration comparing to that obtained prior to an optimization.

The Laccase Engineering Database: a classification and analysis system for laccases and related multicopper oxidases.

Laccases and their homologues form the protein superfamily of multicopper oxidases (MCO). They catalyze the oxidation of many, particularly phenolic substances, and, besides playing an important role in many cellular activities, are of interest in biotechnological applications. The Laccase Engineering Database (LccED, http://www.lcced.uni-stuttgart.de) was designed to serve as a tool for a systematic sequence-based classification and analysis of the diverse multicopper oxidase protein family. More than 2200 proteins were classified into 11 superfamilies and 56 homologous families. For each family, the LccED provides multiple sequence alignments, phylogenetic trees and family-specific HMM profiles. The integration of structures for 14 different proteins allows a comprehensive comparison of sequences and structures to derive biochemical properties. Among the families, the distribution of the proteins regarding different kingdoms was investigated. The database was applied to perform a comprehensive analysis by MCO- and laccase-specific patterns. The LccED combines information of sequences and structures of MCOs. It serves as a classification tool to assign new proteins to a homologous family and can be applied to investigate sequence-structure-function relationship and to guide protein engineering. Database URL: http://www.lcced.uni-stuttgart.de.

Biocatalytic process optimization based on mechanistic modeling of cholic acid oxidation with cofactor regeneration.

Reduction and oxidation of steroids in the human gut are catalyzed by hydroxysteroid dehydrogenases of microorganisms. For the production of 12-ketochenodeoxycholic acid (12-Keto-CDCA) from cholic acid the biocatalytic application of the 12α-hydroxysteroid dehydrogenase of Clostridium group P, strain C 48-50 (HSDH) is an alternative to chemical synthesis. However, due to the intensive costs the necessary cofactor (NADP(+) ) has to be regenerated. The alcohol dehydrogenase of Thermoanaerobacter ethanolicus (ADH-TE) was applied to catalyze the reduction of acetone while regenerating NADP(+) . A mechanistic kinetic model was developed for the process development of cholic acid oxidation using HSDH and ADH-TE. The process model was derived by identifying the parameters for both enzymatic models separately using progress curve measurements of batch processes over a broad range of concentrations and considering the underlying ordered bi-bi mechanism. Both independently derived kinetic models were coupled via mass balances to predict the production of 12-Keto-CDCA with HSDH and integrated cofactor regeneration with ADH-TE and acetone as co-substrate. The prediction of the derived model was suitable to describe the dynamics of the preparative 12-Keto-CDCA batch production with different initial reactant and enzyme concentrations. These datasets were used again for parameter identification. This led to a combined model which excellently described the reaction dynamics of biocatalytic batch processes over broad concentration ranges. Based on the identified process model batch process optimization was successfully performed in silico to minimize enzyme costs. By using 0.1 mM NADP(+) the HSDH concentration can be reduced to 3-4 µM and the ADH concentration to 0.4-0.6 µM to reach the maximal possible conversion of 100 mM cholic acid within 48 h. In conclusion, the identified mechanistic model offers a powerful tool for a cost-efficient process design.

Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7ß-hydroxysteroid dehydrogenase from Collinsella aerofaciens.

A gene encoding an NADPH-dependent 7ß-hydroxysteroid dehydrogenase (7ß-HSDH) from Collinsella aerofaciens DSM 3979 (ATCC 25986, formerly Eubacterium aerofaciens) was identified and cloned in this study. Sequence comparison of the translated amino acid sequence suggests that the enzyme belongs to the short-chain dehydrogenase superfamily. This enzyme was expressed in Escherichia coli with a yield of 330 mg (5,828 U) per liter of culture. The enzyme catalyzes both the oxidation of ursodeoxycholic acid (UDA) forming 7-keto-lithocholic acid (KLA) and the reduction of KLA forming UDA acid in the presence of NADP(+) or NADPH, respectively. In the presence of NADPH, 7ß-HSDH can also reduce dehydrocholic acid. SDS-PAGE and gel filtration of the expressed and purified enzyme revealed a dimeric nature of 7ß-HSDH with a size of 30 kDa for each subunit. If used for the oxidation of UDA, its pH optimum is between 9 and 10 whereas for the reduction of KLA and dehydrocholic acid it shows an optimum between pH 4 to 6. Usage of the enzyme for the biotransformation of KLA in a 0.5-g scale showed that this 7ß-HSDH is a useful biocatalyst for producing UDA from suitable precursors in a preparative scale.

Biosynthesis of zeaxanthin in recombinant Pseudomonas putida.

Pseudomonas putida KT2440 strain was investigated for biosynthesis of the valuable xanthophyll zeaxanthin. A new plasmid was constructed harboring five carotenogenic genes from Pantoea ananatis and three genes from Escherichia coli under control of an L: -rhamnose-inducible promoter. Pseudomonas putida KT2440 wild type hardly tolerated the plasmids for carotenoid production. Mating experiments with E. coli S17-1 strains revealed that the carotenoid products are toxic to the Pseudomonas putida cells. Several carotenoid-tolerant transposon mutants could be isolated, and different gene targets for relief of carotenoid toxicity were identified. After optimization of cultivation conditions and product processing, 51 mg/l zeaxanthin could be produced, corresponding to a product yield of 7 mg zeaxanthin per gram cell dry weight. The effect of various additives on production of hydrophobic zeaxanthin was investigated as well. Particularly, the addition of lecithin during cell cultivation increased volumetric productivity of Pseudomonas putida by a factor of 4.7 (51 mg/l vs. 239 mg/l).

Analysis of phosphorothionate pesticides using a chloroperoxidase pretreatment and acetylcholinesterase biosensor detection.

Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the nervous system. It is inhibited by organophosphate and carbamate pesticides. However, this enzyme is only slightly inhibited by organophosphorothionates, which makes the detection of these pesticides analytically very difficult. A new enzymatic method for the activation and detection of phosphorothionates was developed with the capability to be used directly in food samples without the need of laborious solvent extraction steps. Chloroperoxidase (CPO) from Caldariomyces fumago was combined with tert-butyl hydroperoxide and two halides. Chlorpyrifos and triazophos were completely oxidized. Fenitrothion, methidathion and parathion methyl showed conversion rates between 54 and 61%. Furthermore, the oxidized solution was submitted to an AChE biosensor assay. Chlorpyrifos spiked in organic orange juice was oxidized, where its oxon product was detected in concentrations down to 5 microg/L (final concentration food sample: 25 microg/L). The complete duration of the method takes about 2 h.

Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola.

The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40 degrees C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 +/- 160 U mg(-1)) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.

Rational design of Pseudozyma antarctica lipase B yielding a general esterification catalyst.

Pseudozyma antarctica lipase B (CALB) shows activity in the acrylation of hydroxypropylcarbamate, a racemic mixture of enantiomers of primary and secondary alcohols. However, full conversion is hampered by the slowly reacting S enantiomer of the secondary alcohol. The same is true for a wide range of secondary alcohols, for example, octan-2- and -3-ol. In order to get high conversion in these reactions in a short time, the stereospecificity pocket of CALB was redesigned by using predictions from molecular modeling. Positions 278, 104, and 47 were targeted, and a library for two-site saturation mutagenesis at positions 104 and 278 was constructed. The library was then screened for hydrolysis of acrylated hydroxypropylcarbamates. The best mutants L278A, L278V, L278A/W104F, and L278A/W104F/S47A showed an increased conversion in hydrolysis and transesterification of more than 30 %. While the wild-type showed only 73 % conversion in the acrylation of hydroxypropylcarbamate after 6 h, 97 % conversion was achieved by L278A in this time. Besides this, L278A/W104F reached >96 % conversion in the acrylation of octan-2- and -3-ol within 48 h and showed a significant decrease in stereoselectivity, while the wild-type reached only 68 and 59 % conversion, respectively. Thus the new biocatalysts can be used for efficient transformation of racemic alcohols and esters with high activity when the high stereoselectivity of the wild-type hampers complete conversion of racemic substrates in a short time.

Engineering cytochrome P450 monooxygenase CYP 116B3 for high dealkylation activity.

Cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber catalyzes the dealkylation of 7-ethoxycoumarin and the hydroxylation of substituted and unsubstituted aromatics. However, since activities were quite low, a combination of site-specific mutagenesis and directed evolution was applied to produce 7800 variants of CYP116B3, which were screened via a newly developed high-throughput screening system based on the dealkylation of 7-ethoxycoumarin catalyzed by recombinant E. coli. The best mutant was found after four rounds of directed evolution and had a 240-fold increased deethylation activity toward 7-ethoxycoumarin (223 nmol product/nmol P450.min) and a 10-fold increased demethylation activity toward 7-methoxycoumarin (9 nmol product/nmol P450.min).

Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes.

Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system.

BACKGROUND: (+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli. RESULTS: Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+)-valencene (1) yielding nootkatol (2 and 3) and (+)-nootkatone (4). However, when the in vivo biooxidation of (+)-valencene (1) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents - isooctane, n-octane, dodecane or hexadecane - were set up, resulting in accumulation of nootkatol (2 and 3) and (+)-nootkatone (4) of up to 97% of the total product. The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane. CONCLUSION: This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up.

Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase.

BACKGROUND: The characteristic of most lipases is the interfacial activation at a lipid interface or in non-polar solvents. Interfacial activation is linked to a large conformational change of a lid, from a closed to an open conformation which makes the active site accessible for substrates. While for many lipases crystal structures of the closed and open conformation have been determined, the pathway of the conformational transition and possible bottlenecks are unknown. Therefore, molecular dynamics simulations of a closed homology model and an open crystal structure of Burkholderia cepacia lipase in water and toluene were performed to investigate the influence of solvents on structure, dynamics, and the conformational transition of the lid. RESULTS: The conformational transition of B. cepacia lipase was dependent on the solvent. In simulations of closed B. cepacia lipase in water no conformational transition was observed, while in three independent simulations of the closed lipase in toluene the lid gradually opened during the first 10-15 ns. The pathway of conformational transition was accessible and a barrier was identified, where a helix prevented the lid from opening to the completely open conformation. The open structure in toluene was stabilized by the formation of hydrogen bonds.In simulations of open lipase in water, the lid closed slowly during 30 ns nearly reaching its position in the closed crystal structure, while a further lid opening compared to the crystal structure was observed in toluene. While the helical structure of the lid was intact during opening in toluene, it partially unfolded upon closing in water. The closing of the lid in water was also observed, when with eight intermediate structures between the closed and the open conformation as derived from the simulations in toluene were taken as starting structures. A hydrophobic beta-hairpin was moving away from the lid in all simulations in water, which was not observed in simulations in toluene. The conformational transition of the lid was not correlated to the motions of the beta-hairpin structure. CONCLUSION: Conformational transitions between the experimentally observed closed and open conformation of the lid were observed by multiple molecular dynamics simulations of B. cepacia lipase. Transitions in both directions occurred without applying restraints or external forces. The opening and closing were driven by the solvent and independent of a bound substrate molecule.

Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis.

BACKGROUND: Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA. RESULTS: A CotA double mutant, K316N/D500G, was constructed by combining random and site-directed mutagenesis. It can be functionally expressed at an 11.4-fold higher level than the wild-type enzyme. In addition, it is able to convert ferulic acid much faster than the wild-type enzyme (21% vs. 14%) and is far more efficient in decolorizing a range of industrial dyes. The investigation of the effects of the mutations K316N and D500G showed that amino acid at position 316 had a major influence on enzyme activity and position 500 had a major influence on the expression of the laccase. CONCLUSION: The constructed double mutant K316N/D500G of the Bacillus licheniformis CotA laccase is an appropriate candidate for biotechnological applications due to its high expression level and high activity in dimerization of phenolic acids and decolorization of industrial dyes.

Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions.

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12-22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of omega-1, omega-2 and omega-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at delta-position (14% and 16%, respectively), but also produced gamma- and beta-hydroxylated products. delta-Hydroxy lauric and gamma-hydroxy lauric acid are important synthons for the production of the corresponding lactones.

Comparative characterization of four laccases from Trametes versicolor concerning phenolic C-C coupling and oxidation of PAHs.

The laccase genes lccalpha, lccbeta, lccgamma and lccdelta encoding four isoenzymes from Trametes versicolor have been cloned and expressed in Pichia pastoris. Biochemical characterization allowed classification of these laccases into two distinct groups: Lccalpha and Lccbeta possessed higher thermal stability, but lower catalytic activity towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) compared to Lccgamma and Lccdelta. Activities of the laccases were quite different as well. Laccase Lccdelta showed highest phenolic C-C coupling activity with sinapic acid, but lowest oxidizing activity towards polycyclic aromatic hydrocarbons (PAHs). Highest activity towards PAHs was observed with Lccbeta. After 72h, more than 80% of fluorene, anthracene, acenaphthene and acenaphthylene were oxidized by Lccbeta in the presence of ABTS. Investigation of the structural basis of the different activities of the laccases demonstrated the impact of positions 164 and 265 in the substrate binding site on oxidation of PAHs.

Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids.

A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The enzyme has a molecular weight of approximately 65 kDa and demonstrates activity towards canonical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants KM and kcat for ABTS were of 6.5+/-0.2 microM and 83 s(-1), for SGZ of 4.3+/-0.2 microM and 100 s(-1), and for 2,6-DMP of 56.7+/-1.0 microM and 28 s(-1). Highest oxidizing activity towards ABTS was obtained at 85 degrees C. However, after 1 h incubation of CotA at 70 degrees C and 80 degrees C, a residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid by CotA was observed, and highest activity of CotA was found towards sinapic acid.

Identification and functional expression of a Delta9-fatty acid desaturase from Psychrobacter urativorans in Escherichia coli.

The Delta9-fatty acid desaturase is a key enzyme in the synthesis of unsaturated fatty acids. The fatty acid composition of membrane phospholipids in Psychrobacter urativorans is characterized by a high degree of desaturation at Delta9 position. Based on CODEHOP-mediated PCR strategy, a novel gene designated as PuFAD9, putatively encoding a Delta9-fatty acid desaturase (PuFAD9), was isolated from P. urativorans. The gene consists of 1,455 bp and codes for 484 amino acids. Analysis of the amino acid sequence reveals three histidine clusters and a hydropathy profile, typical for membrane-bound desaturases. Activity of the PuFAD9 protein, recombinantly expressed in Escherichia coli was confirmed by GC-MS analysis of the cellular fatty acid composition. It was found that the ratio between palmitoleic and palmitic acid in E. coli cells heterologously expressing the PuFAD9 gene was significantly affected by IPTG induction and the growth temperature.