Preparation of Chiral Hydroxy Esters Using Actinobacteria : Biocatalyst Activity of Marine-Derived Micromonospora and Streptomyces Strains

To research the potential ability of marine-derived actinomycetes to act as biocatalysts, 8 Micromonospora strains and 5 Streptomyces strains were screened. Two recommended media (227 and 1076 media) and 2 modified media (1076-25% and P-1076-25% media) for liquid culture of these marine-derived actinomycetes were tested. As a result, 2 Micromonospora strains (Micromonospora sp. NBRC107096 and 107097) cultured with the 1076-25% medium and 2 Streptomyces strains (Streptomyces tateyamensis NBRC105048 and Streptomyces sp. NBRC105896) cultured with P-1076-25% medium showed a good growth. The stereoselective reduction of α-keto esters using these 4 actinomycetes was tested. As a result, it was found that these strains had a reducing activity toward various α-keto esters. The introduction of L-glutamate or sucrose as an additive remarkably increased the conversion ratios in the reduction of substrates by the Micromonospora strain. Furthermore, in the presence of L-alanine, Streptomyces tateyamensis NBRC105048 reduced ethyl pyruvate, ethyl 2-oxobutanoate, ethyl 2-oxopentanoate, ethyl 2-oxohexanoate, and ethyl 3-methyl-2-oxobutyrate to the corresponding α-hydroxy ester with a high conversion ratio and with excellent enantiomeric excess. Thus, we found that these marine-derived actinomycetes have great potential to be used as biocatalysts for stereoselective reduction of carbonyl compounds.

Many actinomycetes have been isolated and screened from soil in the last few decades [15].Recently, the rate of novel metabolites discovery from terrestrial actionmycetes has decreased significantly [16], therefore the search for marine actinomycetes, including free-living and marine invertebrate-associated actinomycetes, has attracted attention as an alternative approach [17][18][19][20].For example, it was found that strains of Salinispora-marine actinomycetes-produce useful bioactive compounds such as arenimycin and salinosporamide A [21,22].Moreover, it has been reported that Salinispora strains reduce toward α-keto esters and their derivatives stereoselectively [23].Thus, new applications of marine actinomycetes are expected from the viewpoint of asymmetric syntheses of bioactive substances.However, the potential biocatalyst activity of other marine-derived actinomycetes has not been investigated.

Mreduction of α-Keto Esters with Marine Actinomycetes Whole Cells
Saline-washed wet cells (0.5 g, dry weight approximately 0.2 g) were resuspended in a large test tube ( 30 mm × 200 mm) containing 20 mL of saline.The substrate (0.15 mmol; corresponding substrate concentration was 7.5 mM) and additive (5.0 mmol) were added, and the reaction mixture was incubated aerobically (reciprocating shaking at 120 min -1 ) at 25˚C.A portion (0.5 mL) of the mixture was filtered using a short diatomaceous earth column ( 10 mm × 30 mm), extracted with diethyl ether (5.0 mL), and then concentrated under reduced pressure.

Microorganisms and Culture
Conversions of the produced alcohols (Figure The enantiomeric excess (e.e.) of the product was measured using a GLC equipped with an optically active CP-Chirasil-DEX CB (2a-e, 2g-h) or Gamma DEX 225 capillary column (2f).The e.e. was calculated according to the following formula: e.e. (%) = {(R -S)/(R + S)} × 100.These R and S are the respective peak areas on GLC analyses.The absolute configurations of α-hydroxy esters (2a-h) were identified by comparing their retention times from the GLC analyses with those of authentic samples [24].

Screening of Marine-Derived Actinomycetes and Culture Media
To search for a suitable medium for the liquid culture, the amount of wet cells obtained by cultivating of marine actinomycetes in various culture media was measured.
The NBRC-recommended medium for 8 Micromonospora strains tested in this study was the 227 medium.Micromonospora strains cultured in the 227 medium, with the exception of NBRC 107094 and 107095, produced 5.0 g or less of the wet cells (Table 1).To improve the culture rate in the 227 medium, 3 new culture media (1076, 1076-25%, and P-1076-25% medium) containing marine water were tested for efficacy (1076-25% and P-1076-25% media were newly designed in this study).
In the cultivation of these 8 strains using the 1076 medium including marine water (50% concentration), although over 6.0 g of wet cells of the NBRC107096 strain were obtained, 2.0 g or less of wet cells were obtain-ed for the NBRC107093, 107095, 107097 and 107098 strains.On the other hand, we obtained over 7.0 g of wetcells when the NBRC107096 and 107097 strains were cultured in the 1076-25% medium (25% marine water).Furthermore, to obtain more wet cells, Micromonospora strains were cultured in P-1076-25% medium including polypepton (25% marine water); however, 5.0 g of wet cells was not obtained.
In a similar manner, a suitable medium for the liquid culture of 5 marine-derived Streptomyces strains was determined (Table 2).We found that the P-1076-25% medium is most suitable for the liquid culture of the Streptomyces strains; in particular, the culture of Streptomyces sp. and S. tateyamensis yielded over 5.0 g of wet cells.In contrast, Streptomyces marinus did not show good growth in any culture medium (less than 1.0 g of wet cells was obtained).
Therefore, we investigated the possibility that 2 Micromonospora strains (NBRC107096 and 107097) and 2 Streptomyces strains (Streptomyces tateyamensis NBRC105048 and Streptomyces sp.NBRC105896) can act as biocatalysts for the asymmetric reduction of carbonyl compounds.

Reduction of α-Keto Esters by Marine-Derived Micromonospora Strains
Two Micromonospora strains (NBRC107096 and 107097) were tested for their ability to reduce α-keto esters (Figure 1).The results of the α-keto ester reductions are summarized in Table 3.We found that both Micromonospora strains reduced aliphatic and aromatic α-keto esters (1a-h) to the corresponding alcohols (2a-h).The reduction of α-keto esters by the NBRC107097 strain exhibited a higher conversion ratio than the reduction by the NBRC107096 strain; in particular, the NBRC107097   strain had high reducing activity for substrates that had a short alkyl chain.However, except for ethyl pyruvate (1a), the stereoselectivity of the produced alcohols was not high (13% -80% e.e.).In the microbial reduction of carbonyl compounds using bakers' yeast or filamentous fungi (eukaryote), it is well known that the introduction of small organic molecules or metal ions will increase the stereoselectivity of the produced alcohols [25][26][27].In contrast, in the reduction using actinomycetes (prokaryote), there are several reports that the addition of amino acids or sugars is effective in improving the conversion rate and stereoselectivity of products [12][13][14]23].
Therefore, the effect of additives on the reduction of keto esters using marine actinomycetes was investigated (see Table 4).Among various additives (e.g., sugars such as glucose, fructose, sucrose, and maltose or amino acids such as L-alanine, L-glycine, L-glutamate, and Laspartate), the introduction of L-glutamate or sucrose remarkably increased the conversion ratio of the reduc-tion.In particular, the reduction by the NBRC107096 strain in the presence of L-glutamate gave the corresponding α-hydroxy ester with >99% conversion ratio for all substrates.It appears that the increase in reduced nicotinamide-adenine dinucleotide (possibly NADPH) through the oxidative degradation of L-glutamate accelerates the reduction of α-keto esters to the corresponding alcohols.

Reduction of α-Keto Esters by Marine-Derived Streptomyces Strains
Two Streptomyces strains (NBRC105048 and 105896) were tested for their ability to reduce α-keto esters.As shown in Table 3, we found that α-keto esters (1a-h) were reduced by both Streptomyces strains to the corresponding hydroxy esters (2a-h).The NBRC105,896 strain had high reducing activity for α-keto esters when compared with the NBRC105,048 strain and showed excellent conversion ratios (>99% in 2a-d and 2f-h).Fur-thermore, the reduction of 1a-c using the NBRC105,896 strain gave the corresponding (S)-hydroxy esters exclusively (>99% e.e.).An additive was introduced to the reaction mixture to improve the conversion ratio and stereoselectivity (see Table 5).As a result, the reduction of substrates using the NBRC105,048 strain in the presence of L-alanine or sucrose produced the corresponding alcohols with high conversion ratios (L-alanine, >99%, in 2a-d, 2f; sucrose, >99%, in 2a-g).In particular, the introduction of Lalanine improved not only the conversion rate but also the stereoselectivity of the products (>99% e.e. in 2a-f).The effects of other additives (L-glycine, L-glutamate, L-aspartate glucose, fructose, and maltose) were tested; however, the stereoselectivity of the produced alcohols did not increase (data not shown).In contrast, in the reduction by the NBRC105,896 strain, the conversion ratio was decreased by the introduction of the additive.This decrease in the conversion ratio was not expected.

Conclusion
Various α-keto esters were converted to the corresponding α-hydroxy esters by marine-derived actinomycetes.On the basis of the reduction conversion rates and the enantioselectivity of the products, we suggest that Strep-tomyces tateyamensis NBRC105048 and Micromonospora sp.NBRC107096 are potential biocatalysts for the stereoselective reduction of keto esters to obtain chiral hydroxy esters.