Abstract
BackgroundBacillus licheniformis MW3 as a GRAS and thermophilic strain is a promising microorganism for chemical and biofuel production. However, its capacity to co-utilize glucose and xylose, the major sugars found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, a “dual-channel” process was implemented to engineer strain MW3 for simultaneous utilization of glucose and xylose, using l-lactic acid as a target product.ResultsA non-phosphotransferase system (PTS) glucose uptake route was activated via deletion of the glucose transporter gene ptsG and introduction of the galactose permease gene galP. After replacing the promoter of glucokinase gene glck with the strong promoter Pals, the engineered strain recovered glucose consumption and utilized glucose and xylose simultaneously. Meanwhile, to improve the consumption rate of xylose in this strain, several measures were undertaken, such as relieving the regulation of the xylose repressor XylR, reducing the catabolite-responsive element, and optimizing the rate-limiting step. Knockout of ethanol and acetic acid pathway genes further increased lactic acid yield by 6.2%. The resultant strain, RH15, was capable of producing 121.9 g/L l-lactic acid at high yield (95.3%) after 40 h of fermentation from a mixture of glucose and xylose. When a lignocellulosic hydrolysate was used as the substrate, 99.3 g/L l-lactic acid was produced within 40 h, with a specific productivity of 2.48 g/[L h] and a yield of 94.6%.ConclusionsOur engineered strain B. licheniformis RH15 could thermophilically produced l-lactic acid from lignocellulosic hydrolysate with relatively high concentration and productivity at levels that were competitive with most reported cases of l-lactic acid-producers. Thus, the engineered strain might be used as a platform for the production of other chemicals. In addition to engineering the B. licheniformis strain, the “dual-channel” process might serve as an alternative method for engineering a variety of other strains.
Highlights
Bacillus licheniformis MW3 as a generally regarded as safe (GRAS) and thermophilic strain is a promising microorganism for chemical and biofuel production
We evaluated the derivative strain (MW3) [20] of B. licheniformis ATCC 14,580 for its capacity to co-utilize glucose and xylose without carbon catabolite repression (CCR) by producing l-lactic acid, a monomer used to form biodegradable polylactic acid (PLA), as an example
The constituent metabolic engineering strategies have been demonstrated for enhancing glucose and/or xylose utilization in strains such as Corynebacterium glutamicum [30], Enterobacter cloacae [1], Saccharomyces cerevisiae [28], and B. subtilis [27], they have not been used for enhancing l-lactic acid production from glucose and xylose, nor have they been studied in B. licheniformis previously
Summary
Bacillus licheniformis MW3 as a GRAS and thermophilic strain is a promising microorganism for chemical and biofuel production. The production of several biofuels and chemicals, such as ethanol, isobutanol, n-butanol, butane2,3-diol (2,3-BD), and acetone, has been achieved from lignocellulose, most bacteria and yeasts cannot efficiently utilize glucose and xylose simultaneously due to the socalled carbon catabolite repression (CCR) [3, 4]. Via repressing the consumption of other sugars such as xylose, resulting in low efficiency during mixed-sugar fermentation processes [5]. Other reasons for such inefficient processes include the lack of robust genes involved in xylose metabolism or use of the hetero-phosphoketolase pathway, which leads to the generation of equal amounts of byproducts (mainly formic acid, acetic acid, and ethanol) [6]. In an effort to improve the efficiency of this process, growing attention has been devoted to engineering new strains capable of simultaneously utilizing multiple sugars
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