Abstract
Glucose, xylose and arabinose are the three most abundant monosaccharide found in lignocellulosic biomass. Effectively and simultaneously utilization of these sugars by microorganisms for production of the biofuels and bio-chemicals is essential toward directly fermentation of the lignocellulosic biomass. In our previous study, the recombinant Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain was already shown to efficiently utilize xylose for production of acetoin, with a yield of 0.36 g/g xylose. In the current study, the Bacillus subtilis168ARSRCPΔacoAΔbdhA strain was further engineered to produce acetoin from a glucose, xylose, and arabinose mixtures. To accomplish this, the endogenous xylose transport protein AraE, the exogenous xylose isomerase gene xylA and the xylulokinase gene xylB from E. coli were co-overexpressed in the Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain, which enabled the resulting strain, denoted ZB02, to simultaneously utilize glucose and xylose. Unexpectedly, the ZB02 strain could simultaneously utilize glucose and arabinose also. Further results indicated that the transcriptional inhibition of the arabinose transport protein gene araE was the main limiting factor for arabinose utilization in the presence of glucose. Additionally, the arabinose operon in B. subtilis could be activated by the addition of arabinose, even in the presence of glucose. Through fed-batch fermentation, strain ZB02 could simultaneously utilize glucose, xylose, and arabinose, with an average sugar consumption rate of 3.00 g/l/h and an average production of 62.2 g/l acetoin at a rate of 0.864 g/l/h. Finally, the strain produced 11.2 g/l acetoin from lignocellulosic hydrolysate (containing 20.6g/l glucose, 12.1 g/l xylose and 0.45 g/l arabinose) in flask cultivation, with an acetoin yield of 0.34 g/g total sugar. The result demonstrates that this strain has good potential for the utilization of lignocellulosic hydrolysate for production of acetoin.
Highlights
Acetoin, known as 3-hydroxy-2-butanone, is widely used as a flavoring agent in the food industry and as an industrial raw material and precursor in the synthesis of various important compounds [1]
The B. subtilis mutant TH-49 was obtained by treating the B. subtilis strain NT-50-44 with UV irradiation and NTG (Nitroso-guanidine) mutagenesis, and its acetoin production rate reached 56.9 g/l when grown in a 100-l fermenter in the presence of glucose [15]
As B. subtilis 168 cannot utilize xylose as a single carbon source, we developed strain 168ARSRCPΔacoAΔbdhA in a previous study [30]. This strain harbors three beneficial point mutations that enable B. subtilis to efficiently utilize xylose to produce acetoin. Xylose utilization in this strain is still subject to glucose repression: when xylose and glucose are both present in the medium, strain Bacillus subtilis 168ARSRCPΔacoAΔbdhA can not consume the xylose until the glucose is depleted
Summary
Known as 3-hydroxy-2-butanone, is widely used as a flavoring agent in the food industry and as an industrial raw material and precursor in the synthesis of various important compounds [1]. Acetoin can be produced through three methods: chemical synthesis, enzymatic conversion and microbial fermentation [3]. Among these methods, the microbial fermentation production of acetoin is the most costeffective and environment friendly strategy [4]. The most widely used methods to obtain strains that produce high yields of acetoin are the screening of natural populations and the physical or chemical mutation [6], [12,13,14]. Sun et al [4] overexpressed NADH oxidase in the Serratia marcescens H32 strain, and the final acetoin titer was improved by 33% to 75.2 g/l, the highest level of acetoin production obtained by fermentation to date. Developing a bioprocess capable of high levels of acetoin production based on the fermentation of pentose, the second most abundant sugar in lignocellulosic hydrolysate, is a promising method for further reducing the associated production costs [19]
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