By genome sequencing and analysis, we have been able to identify a gene cluster encoding a four component oxygenase which is able to oxidize d-limonene, l-limonene, cumene, and indole. Heterologous expression of the complete oxygenase system in Pseudomonas putida S12 allowed efficient and highly regiospecific biotransformation of d-limonene into (+)-trans-carveol. The discovered oxygenase represents a promising biocatalyst for the biotechnological valorisation of d-limonene.

Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format. For this, we first developed an expression system for production of phenylacetone monooxygenase (PAMO) in the periplasm of E. coli. This allows probing the enzyme for any target substrate while it is also compatible with extracellular coenzyme regeneration. For coenzyme regeneration, we used phosphite dehydrogenase, which forms phosphate upon NADPH recycling. This allowed the use of a chromogenic molybdate-based phosphate determination assay. The screening procedure was supplemented with a detection method for identification of mutant enzymes that act as NADPH oxidases, thereby excluding false-positives. The whole-cell–based screening method was validated by screening site–saturation libraries of PAMO and resulted in the identification of PAMO mutants with altered catalytic properties. This new method can be used for screening libraries of BVMOs for activity with any desired substrate and therefore is a powerful tool for engineering of these enzymes.

Background: Baeyer-Villiger monooxygenases (BVMOs) represent a group of enzymes of considerable biotechnological relevance as illustrated by their growing use as biocatalyst in a variety of synthetic applications. However, due to their increased use the reproducible expression of BVMOs and other biotechnologically relevant enzymes has become a pressing matter while knowledge about the factors governing their reproducible expression is scattered.Results: Here, we have used phenylacetone monooxygenase (PAMO) from Thermobifida fusca, a prototype Type I BVMO, as a model enzyme to develop a stepwise strategy to optimize the biotransformation performance of recombinant E. coli expressing PAMO in 96-well microtiter plates in a reproducible fashion. Using this system, the best expression conditions of PAMO were investigated first, including different host strains, temperature as well as time and induction period for PAMO expression. This optimized system was used next to improve biotransformation conditions, the PAMO-catalyzed conversion of phenylacetone, by evaluating the best electron donor, substrate concentration, and the temperature and length of biotransformation. Combining all optimized parameters resulted in a more than four-fold enhancement of the biocatalytic performance and, importantly, this was highly reproducible as indicated by the relative standard deviation of 1% for non-washed cells and 3% for washed cells. Furthermore, the optimized procedure was successfully adapted for activity-based mutant screening.Conclusions: Our optimized procedure, which provides a comprehensive overview of the key factors influencing the reproducible expression and performance of a biocatalyst, is expected to form a rational basis for the optimization of miniaturized biotransformations and for the design of novel activity-based screening procedures suitable for BVMOs and other NAD(P)H-dependent enzymes as well.

This chapter mainly talks about microtiter plates (MTPs) and reflects their current importance in industry and academia alike. Analysis of various culture preservation techniques and inoculum development techniques, with special attention to reproducibility of resulting cultures in terms of morphology (in MTPs and flasks), natural product yields and enzyme activity, and growth rates and nutrient utilization, is important. Variations in either inoculum or inoculation conditions may therefore lead to wide-ranging culture morphology changes. Important factors include the scheme employed for populating strains into the miniaturized format, the medium used for synchronous growth among the populations of strains in any one device, appropriate inoculation protocols, optimum strain preservation protocols, and choice of MTP format. Test tubes are used for various purposes by the microbiologist, but the chapter focuses on use of test tubes for high-throughput culturing of microbes in small volumes. Miniaturized cultivation systems are attractive not only in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. Provided the user is willing to invest a sufficient amount of time and effort on the optimization for their specific purpose, the presently available systems allow the cultivation at a 0.1- to 10-ml scale at a reproducibility and quality approaching those of larger-scale fermentation equipment.

ABSTRACT The microbial biotransformation of Δ 9 -tetrahydrocannabinol was investigated using a collection of 206 alkane-degrading strains. Fifteen percent of these strains, mainly gram-positive strains from the genera Rhodococcus , Mycobacterium , Gordonia , and Dietzia , yielded more-polar derivatives. Eight derivatives were produced on a mg scale, isolated, and purified, and their chemical structures were elucidated with the use of liquid chromatography-mass spectrometry, 1 H-nuclear magnetic resonance ( 1 H-NMR), and two-dimensional NMR ( 1 H- 1 H correlation spectroscopy and heteronuclear multiple bond coherence). All eight biotransformation products possessed modified alkyl chains, with hydroxy, carboxy, and ester functionalities. In a number of strains, β-oxidation of the initially formed C 5 carboxylic acid led to the formation of a carboxylic acid lacking two methylene groups.

Laboratory of Ecotoxicology, Nationa I Institute of Public Health and Environmental Protection, PO Box 1, 3720 BA Bilthoven, The Netherlands Previous studies have shown that expression of the toluene and mand p-xylene degradation pathway in Pseudomonas putids ( p W 0 ) is subject to catabolite repression by succinate. We report here that the expression level of the upper part of this so-called TOL pathway in cells grown in chemostat culture is strongly influenced by nutrient limitation when m-xylene is the sole carbon and energy source. The benzylalcohol dehydrogenase (BADH) levels in cells that are growth-limited by anabolic processes [sulphate (S)-, phosphate (P)or nitrogen (N)-limiting conditions] were 3-12O/0 of those in cells growing under oxygen limitation (when catabolism limits growth). BADH levels under S-, Pand N-limitation were further decreased (threeto fivefold) when succinate was supplied in addition to m-xylene. Levels of the meta-cleavage pathway enzyme catechoI2,3=dioxygenase were less affected by the growth conditions but the general pattern was similar. Dilution rate also influenced the expression of the TOL pathway: BADH levels gradually decreased with increasing dilution rates, from 1250 mU (mg protein)'l at D = 095 h'l under m-xylene limitation to 290 mU (mg protein)" a t D = 058 h'l (non-limited growth). BADH levels were shown to be proportional to the specific affinity of whole cells for m-xylene. It may, therefore, be expected that natural degradation rates are adversely affected by anabolic nutrient limitations, especially at relatively low concentrations of the xenobiotic compound.

In the past decade, the use of microtiter plates for microbial growth has become widespread, particularly in industry. In parallel, research in academia has provided a thorough insight into the complex relation between well dimensions, culture volumes, orbital shaking conditions and surface tension on the one hand, and oxygen-transfer rates and degrees of mixing on the other. In this review, I will discuss these fundamental issues and describe the current applications of microtiter plates in microbiology. Microtiter plates can now be considered a mature alternative to Erlenmeyer shake flasks.

ABSTRACT A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (−)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate l -limonene in the 7 position to produce enantiopure (−)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams.

A diverse range of microorganisms capable of growth on phenylacetic acid as the sole source of carbon and energy were isolated from soil. Sixty six different isolates were identified and grouped according to 16S rRNA gene RFLP analysis. Subsequent sequencing of 16S rDNA from selected strains allowed further characterization of the phenylacetic acid degrading population isolated from soil. Nearly half (30) of the isolates are Bacillus species while the rest of the isolates are strains from a variety of genera namely, Arthrobacter, Pseudomonas, Rhodococcus, Acinetobacter, Enterobacter, Flavobacterium, and Paenibacillus. Sixty-one of the sixty-six strains reproducibly grew in defined minimal liquid culture medium (E2). All strains isolated grew when at least one hydroxylated derivative of phenylacetic acid was supplied as the carbon source, while 59 out of the 61 strains tested, accumulated ortho-hydroxyphenylacetic acid in the assay buffer; when pulsed with phenylacetic acid. Oxygen consumption experiments failed to indicate a clear link between phenylacetic acid and hydroxy-substituted phenylacetic acid in isolates from a broad range of genera.

For Abstract see ChemInform Abstract in Full Text.

Crude cell extracts of Pseudomonas putida F6 transformed 4-substituted fluoro-, chloro-, bromo- and iodo-phenol without the exogenous addition of cofactors. The rate of substrate consumption decreased with increasing substituent size (F>Cl>Br>I). Biotransformations resulted in greater than 95% utilisation of the halogenated substrate. Product accumulation was observed in incubations with 4-chloro, 4-bromo- and 4-iodo-phenol. These products were identified as the corresponding 4-substituted catechols. Transformation of 4-fluorophenol did not result in the accumulation of the corresponding catechol; however, manipulation of the reaction conditions by incorporation of ascorbic acid culminated in the formation of 4-fluorocatechol. Cell extracts of P. putida F6 also showed activity towards a 3-substituted phenol, namely 3-fluorophenol, resulting in the formation of a single product, 4-fluorocatechol.

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (alpha-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.

ABSTRACT Pseudomonas putida F6 was found to metabolize p -hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p -hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage.

The styrene degradative pathway in Pseudmonas putida CA-3 has previously been shown to be divided into an upper pathway involving the conversion of styrene to phenylacetic acid and a lower pathway for the subsequent degradation of phenylacetic acid. It is reported here that expression of the regulatory genes styS and styR is essential for transcription of the upper pathway, but not for degradation of the lower pathway inducer, phenylacetic acid. The presence of phenylacetic acid in the growth medium completely repressed the upper pathway enzymes even in the presence of styrene, the upper pathway inducer. This repression is mediated at the transcription level by preventing expression of the styS and styR regulatory genes. Finally, an examination was made of the various stages of the diauxic growth curve obtained when P. putida CA-3 was grown on styrene together with an additional carbon source and it is reported that catabolite repression may involve a different mechanism to transcriptional repression by an additional carbon source.

Mycelium-forming Streptomyces strains were grown in one milliliter liquid micro-cultures in square deep-well microtiter plates. Growth was evaluated with respect to biomass formation and production of secondary metabolites which were found to be very similar in the micro-cultures, bioreactor, and shake flask cultivations, respectively. Despite repetitive sampling and extensive growth on the walls of the wells, no cross contamination occurred. Furthermore, we successfully employed cold storage at −20 °C of spore suspensions (in the 96–well format), directly prepared from cultures grown on agar in the microtitre plate. Cultures were retrieved by replicating aliquots from the frozen spore suspensions.