Conversion by Freshly Cultured Cells
Collectively, the “freshly” grown microbes converted the various ketone substrates to different degrees, with some differences observed between complex and defined media (Figures 2A-D). Conversion of a ketone substrate is a function of the selectivity of one or more enzymes within the cell but also the relative expression of those enzymes that can be impacted by growth media composition. In most cases, the biocatalytic activity in complex media was greater or equivalent to that observed in defined media with the noted exception of C. parapsilosis (ATCC® 22019™), which was able to convert ethyl acetoacetate only under defined media conditions. However, conversion based on growth in complex media increased the number of background peaks on the chromatograms, perhaps making the defined media more desirable for interpreting data for other substrates (data not shown). Other growth parameters such as temperature, pH, or primary carbon source were not explored as part of this study, although these variables can still have an impact on the extent and rate of conversion.
Interestingly, there were differences across the entire group of strains in the conversion of ethyl acetoacetate (Figure 2A) vs. the chlorinated analogue ethyl 4-chloroacetoacetate (Figure 2B). This suggests that the keto-reductases within some of the organisms can tolerate or even prefer an electron withdrawing group. None of the strains were particularly prolific at converting acetophenone to phenylethanol, though specificity of the enzymes and expression level cannot be differentiated. No attempt was made to measure enantiomeric excess (%ee) as it is generally accepted that microbes, in particular yeasts, can have more than one enzyme that can reduce ketones and the resulting %ee is a composite of these different enzymes with different stereospecificities.
Figure 2. Conversion of (A) ethyl acetoacetate, (B) ethyl 4-chloroacetoacetate, (C) ethyl pyruvate, and (D) acetophenone to their corresponding
alcohols by ATCC strains freshly grown and resuspended in complex or defined media.
Conversion by Lyophilized Cells Used Directly
The use of lyophilized cells allows the growth and preparation of the microbial catalyst to be temporally separated from the actual screening. Moreover, microbial catalysts can be lyophilized in a well-plate format and subsequently screened in an automated fashion along with traditional chemical catalysts. Encouraging results were obtained for the use of lyophilized microbial catalysts stored at 4°C, without a period of outgrowth after resuspension and prior to substrate addition, and suggest that a lyophilized keto-reductase “panel” can be created in deep-well plates to facilitate rapid screening without the need to carry out culturing at the time of bioconversion. Conversion capabilities for the lyophilized strains were generally reduced, and in some cases eliminated, compared to that of actively metabolizing strains (Figures 3A-D). However, there were some instances where lyophilization and direct use improved substrate conversion – e.g., C. parapsilosis (ATCC® 22019™) / ethyl acetoacetate and S. venezuelae (ATCC® 15439™ / ethyl pyruvate).
The use of 10% (w/v) trehalose is a generally accepted lyoprotectant, but others along with different growth conditions and the physiological state of the cells at the time of lyophilization are known to impact viability17 and by extension, the ability to convert ketone substrates. Moreover, the impact of different preservation variables varies by strain. This is particularly useful for identifying a strain possessing a keto-reductase enzyme active against a substrate of interest that ultimately will be expressed in a heterologous host, and where the absolute conversion capability of the native strain is not as important.
Figure 3. Conversion of (A) ethyl acetoacetate, (B) ethyl 4-chloroacetoacetate, (C) ethyl pyruvate and (D) acetophenone to their corresponding alcohols by ATCC strains lyophilized in complex or defined media plus 10% (w/v) trehalose, stored at 4°C.
Keto-reductase Diversity of Sequenced Strains
S. cerevisiae possesses several enzymes from different superfamilies capable of carrying out the reduction of ketone substrates.4 Analysis of whole-genome sequence data from 19 of the yeast strains were analyzed for keto-reductase diversity based on aldo-keto reductase (AKR) and short chain dehydrogenase/reductase (SDR) enzyme superfamilies using Prosite18 and Pfam19 motifs. Based on the presence of at least one Prosite or Pfam signature, these 19 strains possess a total of approximately 345 AKRs and 547 SDRs (892 total) that are potentially capable of converting ketone substrates to alcohols (Figure 4). K. lactis (ATCC® 8585™) had the most AKRs and SDRs of the group with a total of 87. The Candida genus has long been a source of keto-reductase enzymes and in this study; sequencing revealed 73 total AKRs and SDRs for C. guilliermondii (ATCC® 6260™) while C. krusei (ATCC® 34135™) and C. tropicalis (ATCC® 13803™) possess a total of 60 and 64 respectively. Surprisingly L. elongisporus (ATCC® 11503™) possessed 78 total enzymes, and there has been one report of an AKR from this strain converting ethyl 4-chloroacetoacetate to the corresponding hydroxybutanoate having been cloned and over-expressed in E. coli.20 S. stipitis (ATCC® 58785™), known for its ability to ferment xylose to ethanol, also represents a rich source of AKR and SDR enzymes with a total of 68 enzymes. By extrapolation, these results suggest that the entire panel of strains may possess over one thousand potential keto-reductases. While duplication of enzymes is very likely, this simple analysis implies an extensive amount of keto-reductase enzyme diversity to be explored for conversion of novel substrates.
Figure 4. Identification of aldo-keto reductase (AKR) and short chain dehydrogenase/reductase (SDR) enzyme superfamily members in whole-genome sequenced yeast strains. *Based on GenBank Reference GCA_000142805.1