Abstract
Backgroundl-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to d-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as d-glucose, leading to a preferential utilization of d-glucose over pentoses. In order to simultaneously utilize both d-glucose and l-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis.ResultsCorynebacterium glutamicum ATCC 31831 utilized d-glucose and l-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized d-glucose over l-arabinose at a high concentration (15 g/L each), although l-arabinose and d-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for d-glucose and l-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of l-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of d-glucose and l-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized d-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of l-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of d-glucose and l-arabinose.ConclusionsMetabolome analysis identified potential bottlenecks in d-glucose and l-arabinose metabolism and was then applied to the following rational metabolic engineering. Manipulation of only two genes enabled simultaneous utilization of d-glucose and l-arabinose at the same rate in metabolically engineered C. glutamicum. This is the first report of rational metabolic design and engineering for simultaneous hexose and pentose utilization without inactivating the phosphotransferase system.
Highlights
Background lArabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to d-xylose
Simultaneous utilization of two sugars was suppressed in the presence of excess glucose Before mixed sugar utilization, sugar consumption and cell growth were investigated in mineral salt medium containing either d-glucose or l-arabinose as sole carbon source (15 g/L) in order to measure the rate of sugar consumption in the absence of a second carbon source
Wild-type strain ATCC 31831 was found to have an equivalent capacity for both l-arabinose metabolism and d-glucose metabolism in the absence of the second carbon source. l-Arabinose was consumed at a rate about equal to that of d-glucose (0.180 and 0.167 g/h/g dry cell weight (DCW), respectively), and the specific growth rate was higher on d-glucose than on l-arabinose [specific growth rate (μ) = 0.40 and 0.36 h−1, respectively] (Fig. 2a)
Summary
Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to d-xylose. Lignocellulosic feedstocks contain cellulose and hemicellulose (14.3–49.9 and 8.8–22.4%, respectively) [1]. Hydrolysis of the lignocellulosic feedstocks yields d-glucose, d-xylose, l-arabinose, and other minor sugar. D-xylose (12–23%) and d-glucose (2–24%) are typically abundant components, followed by l-arabinose (2–6%), the composition of these monosaccharides depends on feedstocks and hydrolysis conditions [2,3,4]. The capability of metabolizing these pentoses and d-glucose simultaneously is important for optimal microbial fermentation to more efficiently utilize carbohydrates contained in lignocellulosic feedstocks, as the hydrolysates are predominantly composed of hexose(s) and pentose(s) [7]. Few microorganisms are capable of utilizing pentoses for bio-based production
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