Study of ChiR function in Serratia marcescens and its application for improving 2,3-butanediol from crystal chitin

Abstract

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.

Controlling and co-ordinating chitinase secretion in aSerratia marcescenspopulation

“…A candidate for controlling the regulation of chitinase production and secretion is ChiR (7,9,11). To explore this experimentally, a strain was constructed in which chiR alone was deleted (BC02, as DB10 chiR, Table 1).…”

“…This suggests stochastic fluctuations in cellular ChiR levels may be the sole determinant of chitinase production in S. marcescens, though the existence of additional more elaborate regulatory factors cannot be ruled out. Indeed, ChiR was originally identified in genetic screens focused on screening for chitinase activity (7,9) and was later exploited as a positive regulator of chitinase activity in biotechnological applications (11). Regulation of chitinases is thought to be further influenced by the presence of chitobiose (the GlcNAc disaccharide) (15) and by small regulatory RNAs (16).…”

“…The LTTR family has been found to regulate the expression of a variety of genes involved in metabolism, virulence, quorum sensing and motility. Deletion of the gene encoding chiR in S. marcescens has been reported to result in a drop in extracellular chitinase activity, while supply of extra copies of chiA on a plasmid resulted in a relative increase in transcripts from the chitinase genes (11).…”

Cloning and characterization of a chitinase from Thermobifida fusca reveals Tfu_0580 as a thermostable and acidic endochitinase

“…Recently, a concept of “chitin refinery” was proposed in 2015 aiming at converting the seafood waste into value-added chemicals. Enzymatic hydrolysis of chitin is one of the most efficient approaches for chitin degradation and has been developed into various applications, such as production of N -acetylglucosamine [ 4 ] or N -acetylchitooligosaccharides [ 5 ], bioconversion of chitinolytic biomass to value-added chemicals [ [6] , [7] , [8] ], and bio-control of fungal phytopathogens [ 9 ].…”

“…To date, a number of native chitinolytic organisms have been isolated such as Serratia marcescens [ 10 ], Bacillus circulans [ 11 ], Acinetobacter parvus [ 12 ], and Aeromonas sp [ 13 ]. Their chitinase activities, functionalities, secretion and regulation mechanisms have been widely characterized and investigated [ 8 , [14] , [15] , [16] ]. These organisms can be potentially developed for a chitin-based consolidated bioprocessing due to the complete chitinase expression, regulation and secretion system [ 8 , 17 ].…”