Enhanced 2,3-Butanediol Production by Optimizing Fermentation Conditions and Engineering Klebsiella oxytoca M1 through Overexpression of Acetoin Reductase.

Sukhyeong Cho,Jinwon Lee,Han Min Woo,Yunje Kim,Youngsoon Um,Taeyeon Kim,Chenyu Du

doi:10.1371/journal.pone.0138109

Abstract

Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase.

Highlights

Because the highest yield of 2,3-BDO was achieved using glucose, further study was performed with glucose as a carbon source for 2,3-BDO production
Acetoin concentration increased after 24 hours, implying the reverse conversion of 2,3-BDO to acetoin in the absence of carbon source as reported in the previous studies [21, 22]
The metabolite profile from 90 g/L of glucose is shown in S1 Fig as a representative result presenting no reverse conversion of 2,3-BDO to acetoin

Summary

Introduction

2,3-butanediol (2,3-BDO) is an important chemical intermediate in numerous industrial applications [1, 2]. K. oxytoca has an advantage on downstream processing over K. pneumoniae owing to much less formation of capsular polysaccharide [13] Several strategies such as altering agitation speeds and reducing by-product formation by metabolic engineering have been developed to enhance 2,3-BDO production with K. oxytoca. Combining a two-stage agitation speed control strategy and metabolic engineering to minimize lactic acid and formic acid production was attempted using K. oxytoca KCTC12133BP ΔldhA ΔpflB in fed-batch fermentation and 113 g/L of 2,3-BDO was obtained [16]. Those previous studies have contributed to high 2,3-BDO production by K. oxytoca, further investigation on fermentation conditions and metabolic engineering strategy is required in order to understand the factors affecting 2,3-BDO production and to facilitate the industrial use of K. oxytoca to obtain higher 2,3-BDO production. 2,3-BDO production was remarkably enhanced up to 142 g/L, which is the highest 2,3-BDO concentration obtained by K. oxytoca strains to date

Materials & Methods

Analytical methods

Results and Discussion

N NaOH1

Conclusions