Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

Abstract

Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. crassa sextuple beta-glucosidase (bgl) gene deletion strains when grown on Avicel without exogenous cellulase addition. The sextuple bgl deletion strains expressing one of the three basally transcribed bgl genes are the best cellobiose producers. For most sextuple strains, the multiple bgl gene deletion has no negative effect on the production of other cellulases. The induction of major endoglucanases and exoglucanases on Avicel in most of the sextuple bgl deletions strains was as fast as or faster than that of the wild type, except for strain F4. The best cellobiose producing strain, F5, produced 7.7 g/L of cellobiose from 20 g/L of Avicel in four days and utilized the Avicel as fast as did the wild type (even in the presence of high cellobiose concentration). The cellobiose yield from cellulose was about 48.3%.

Introduction

Lignocellulosic biomass is a renewable, low cost, and abundant source for the production of fuels and chemicals [1], [2], [3]. The major impediment for the large scale production of ethanol and other biofuels and chemicals from cellulosic biomass is the lack of a low cost processing technology. Specifically, pretreatment and the high cost of cellulase remain two of the most expensive processing steps in converting biomass to fermentable sugars [2], [4], [5], [6].

One of the strategies to lower the processing cost of cellulosic ethanol production is through process consolidation. The conventional platform for cellulosic ethanol production include the following unit operations; pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product separation. Various strategies of process consolidation have been proposed in the past thirty years. Simultaneous saccharification and fermentation (SSF) consolidates the enzymatic hydrolysis and fermentation in one step [7], [8]. Consolidated bioprocessing consolidates the cellulase production, enzymatic hydrolysis, and fermentation [2], [9], [10]. In a previous study, we explored a new biological route for fuels and chemicals production, in which the cellulase production and enzymatic hydrolysis are combined in one step and cellobionate was produced as the reactive intermediate for the subsequent fermentation by a genetically engineered fungus. We also demonstrated that both of the hydrolysate of cellobionate—glucose and gluconate—could be fermented to fuels and chemicals by microorganisms possessing the Entner–Doudoroff pathway such as Escherichia coli KO11 [11]. Ethanol and acetate were produced from gluconate at rates which are even sooner than that of glucose [11]. To divert cellulose toward cellobionate production, it is necessary to disable the fungus's ability to produce BGLs so that cellobiose will be accumulating and over-express cellobiose dehydrogenase, which will oxidize cellobiose to cellobionate [11].

In this study, we investigate the production of cellobiose directly from cellulose, using fungi with multiple bgl genes deleted. BGL is an important component of the cellulase enzyme system. Some BGLs are part of cellulase biosynthesis regulation system since they are responsible for formation of inducers for cellulase production [12], [13], [14], [15], [16], [17]. In this study, we investigate how the deletion of multiple bgl genes affects cellulase production, and cellulose conversion in N. crassa and what combinations of bgl gene deletions can lead to maximal cellobiose yield from cellulose. Such knowledge not only is of importance for designing better process for cellobionate production from cellulose, but also can shed light on processes, in which filamentous fungi are directly used, such as a CBP process using a mixed culture containing fungus.

Neurospora crassa is one of the most efficient plant cell-wall degraders and produces the whole spectrum of cellulases and hemicellulase [18]. It is also a model ascomycete that has been intensively studied for more than 70 years [19], [20], [21], [22], [23]. The tools for its genetic manipulation are well accessible. A near-complete single knockout library facilitated many of the genetic studies [20]. The known and operational sexual cycle enables the quick combination of multiple natural or engineered genetic variants using genetic crossing [23]. N. crassa genome contains seven predicted bgl genes [24]. In this study, we constructed a whole library of sextuple bgl deletion strains by genetic crossing starting from single bgl deletion strains and then investigated direct cellobiose production by these N. crassa mutants. The effects of multiple bgl deletion on cellulose conversion and cellulase production were also studied.