Microbial utilization of chitin, a potential renewable biomass feedstock, is being pursued as a means of developing novel consolidated bioprocessing for the production of chemicals. Serratia marcescens is a gram-negative bacterium that is known for its chitinolytic capability and as a native 2,3-butanediol producer. In S. marcescens, ChiR has been suggested to be a positive regulator of chitinase production. In this study, we aim to understand the effect of ChiR in regulating nine chitinase-related genes in S. marcescens Db11 and demonstrate manipulation of chiR as a useful and efficient genetic target to enhance chitin utilization. First, a chiR overexpression (chiROE) strain and a chiR deletion (ΔchiR) strain were generated and characterized in terms of cellular growth, chitinase activity, and total secreted protein. Compared to the wild-type Db11 strain, the S. marcescens chiROE strain showed an increase in chitinase activity (2.14- to 6.31-fold increase). Increased transcriptional expression of chitinase-related genes was measured using real-time PCR, showing 2.12- to 10.93-fold increases. The S. marcescens ΔchiR strain showed decreases in chitinase activity (4.5- to 25-fold decrease), confirming ChiR's role as a positive regulator of chitinase expression. Finally, chiR overexpression was investigated as a means of increasing biochemical production (2,3-butanediol) from crystal chitin. The chiROE strain produced 1.13 ± 0.08 g/L 2,3-butanediol from 2% crystal chitin, a 2.83-fold improvement from the wild-type strain, indicating ChiR is an important and useful genetic engineering target for enhancing chitin utilization in S. marcescens.

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Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.