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

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.

Using a combined oxygen‐supply and substrate‐feeding strategy to improve 2,3‐butanediol production by metabolically engineered Klebsiella oxytoca KMS005

“…Our 2,3‐BD concentration was lower than, but 2,3‐BD yield was superior to, those of other previous reports . They were also higher than some using K. oxytoca as producers .…”

Synthesis of (3R)-acetoin and 2,3-butanediol isomers by metabolically engineered Lactococcus lactis

“…The maximum (3 R )-acetoin, R-BDO and m-BDO yield reached were 0.42, 0.47 and 0.40 g/g lactose with productivities of 0.64, 1.46 and 0.62 g/L·h respectively. The m-BDO productivity was comparable to that obtained using native 2,3-BDO producers like K. oxytoca 38. RWP is a cheap carbon source, and for this study we also added YE as a nitrogen source.…”

The metabolic flux regulation of Klebsiella pneumoniae based on quorum sensing system

“…In general, butanol is produced from carbohydrates in submerged fermentation by Klebsiella spp 24252627, . Enterobacter spp 28293031, .…”

“…To improve 2,3-butanediol production by Klebsiella spp., strain modification by metabolic engineering has attracted a great deal of attention around the world1724252627. For example, Rathnasingh et al .…”

“…reported that 2,3-butanediol production reached 115.0 g/L in fed-batch fermentation with pure glycerol with the construction of a double mutant ( pduC, ldhA) K. oxytoca strain to reduce the formation of 1,3-propanediol and lactic acid24. This group also discovered that if acetoin reductase was overexpressed in K. oxytoca , acetoin accumulation was significantly reduced, and the highest titre of 2,3-butanediol (142.5 g/L) was achieved25.…”