Enhanced Biomass Yield of and Saccharification in Transgenic Tobacco Over-Expressing β-Glucosidase

Abstract

Here, we report an increase in biomass yield and saccharification in transgenic tobacco plants (Nicotiana tabacum L.) overexpressing thermostable β-glucosidase from Thermotoga maritima, BglB, targeted to the chloroplasts and vacuoles. The transgenic tobacco plants showed phenotypic characteristics that were significantly different from those of the wild-type plants. The biomass yield and life cycle (from germination to flowering and harvest) of the transgenic tobacco plants overexpressing BglB were 52% higher and 36% shorter than those of the wild-type tobacco plants, respectively, indicating a change in the genome transcription levels in the transgenic tobacco plants. Saccharification in biomass samples from the transgenic tobacco plants was 92% higher than that in biomass samples from the wild-type tobacco plants. The transgenic tobacco plants required a total investment (US$/year) corresponding to 52.9% of that required for the wild-type tobacco plants, but the total biomass yield (kg/year) of the transgenic tobacco plants was 43% higher than that of the wild-type tobacco plants. This approach could be applied to other plants to increase biomass yields and overproduce β-glucosidase for lignocellulose conversion.

Keywords:

β-glucosidase; transgenic tobacco plant; biomass yield; overexpression; saccharification

1. Introduction

Lignocellulosic biomass, particularly originating from crops, is abundant and available globally. It acts as an excellent source material for the production of biofuels and value-added products [1,2]. In order to develop biorefineries for processing lignocellulosic biomass, several challenges should be overcome, especially reducing enzyme costs and improving lignocellulosic materials to ensure that they can be hydrolyzed more easily. Recently, several studies focusing on plant genetic engineering have aimed to improve the feasibility of biofuel production by reducing the recalcitrant components of plant biomass and increasing the heterologous overexpression of enzymes for the autohydrolysis of cellulose in plants, in order to improve conversion yield [3,4,5]. Another approach is to increase the production of biomass via plant genetic engineering [6,7]. A recent study reported a considerable improvement in biomass production by fixing photosynthetic inefficiencies, making transgenic tobacco plants to more efficiently recaptures the by-products of photosynthesis with less energy loss, leading to approximately 40% higher biomass production than that of wild-type tobacco plants [8]. However, despite some successes, practical application of this technique has not been confirmed.

β-glucosidases belong to a large family of glycoside hydrolases (http://www.cazy.org/fam/GH1.html) and catalyze the hydrolysis of the β-glucosidic bond between two carbohydrate moieties, or between a carbohydrate and an aglucone moiety. In plants, β-glucosidases play important roles in growth and development, including the (1) release of active forms of phytohormones from their inactivated forms (conjugated-phytohormones), (2) formation of intermediates in cell wall lignification, (3) degradation of cell wall of the endosperm during germination, and (4) activation of chemical defense compounds [9,10,11,12]. Recently, β-glucosidases have garnered attention owing to their biotechnological and industrial applications. In particular, several studies on the heterologous overexpression of β-glucosidase genes in plants have reported an interesting phenomenon, that is, the overexpression of β-glucosidases genes results in enhanced growth and development, increased biomass biosynthesis, and improved saccharification. These changes including early flowering and increased biomass, height, internode length, and leaf area, depending on the changes in hormone levels in transgenic plants have been attributed to the hydrolyzing functions of β-glucosidases, which release activated forms of phytohormones from their inactivated forms (conjugated hormones) [6,7,9,13]. However, the underlying mechanisms in the transgenic plants remain unclear, particularly those involved in changes in genome transcription levels [14,15,16]. In contrast, there were no changes in the level of hormones in transgenic plants in a previous study [17], suggesting that the earlier explanations based on changes in phytohormone levels do not fully explain the observed changes in transgenic plants. Therefore, studies that aim to evaluate the mechanisms leading to pronounced changes in transgenic plants are necessary.

Increased saccharification has been reported in transgenic plants overproducing β-glucosidases, and this can be explained by changes in biomass structures due to modifications in the chemical composition (lignin and saccharide content) of transgenic plants compared with those of the control [18,19,20]. However, the mechanisms underlying increased saccharification, especially how a transgenic plant develops after these changes in the biomass structure or how it achieves enhanced biomass production in some cases, as mentioned above, are not completely clear. Our previous studies indicated that thermostable β-glucosidase from Thermotoga maritima (BglB) could be targeted to the organelles (chloroplasts and vacuoles) of transgenic tobacco plants, while retaining its biological function of enzymatic hydrolysis of lignocellulosic biomass. Furthermore, we provided evidence of enhanced growth and development of transgenic tobacco plants [7,21]. As our previous studies produced encouraging results, in this study, we conducted experiments using many generations of transgenic tobacco plants, with BglB targeted to the chloroplasts and vacuoles, to confirm that the pronounced phenotypic changes in transgenic tobacco plants are stable and can be inherited through generations, and that BglB can be successfully produced by tobacco plants. The results of our RNA microarray analysis provide clear evidence of the changes in genome transcription levels in transgenic tobacco plants in comparison with those in wild-type tobacco plants. Thus, our study provides a novel explanation for enhanced phenotype in transgenic tobacco plants. Because the transgenic tobacco plants showed a significant increase in biomass production, a techno–economic assessment was conducted to confirm the advantages of the BglB transgenic tobacco plants. Moreover, expressing BglB in transgenic tobacco plants may be beneficial because such plants have a superior conversion efficiency of lignocellulosic biomass, because of the combination of β-glucosidases with cellulases. Here, we demonstrate that BglB transgenic plants might be suitable for multi-target biorefinery processes, as their beneficial qualities include an increase in lignocellulosic enzymes. The use of biomass from transgenic plants in biorefinery processes is likely to ensure a high enzymatic conversion yield.

Read more: https://www.mdpi.com/2218-273X/10/5/806