Development of a thermophilic coculture for corn fiber conversion to ethanol

Abstract

The fiber in corn kernels, currently unutilized in the corn to ethanol process, represents an opportunity for introduction of cellulose conversion technology. We report here that Clostridium thermocellum can solubilize over 90% of the carbohydrate in autoclaved corn fiber, including its hemicellulose component glucuronoarabinoxylan (GAX). However, Thermoanaerobacterium thermosaccharolyticum or several other described hemicellulose-fermenting thermophilic bacteria can only partially utilize this GAX. We describe the isolation of a previously undescribed organism, Herbinix spp. strain LL1355, from a thermophilic microbiome that can consume 85% of the recalcitrant GAX. We sequence its genome, and based on structural analysis of the GAX, identify six enzymes that hydrolyze GAX linkages. Combinations of up to four enzymes are successfully expressed in T. thermosaccharolyticum. Supplementation with these enzymes allows T. thermosaccharolyticum to consume 78% of the GAX compared to 53% by the parent strain and increases ethanol yield from corn fiber by 24%.

Fig. 1. Structures of the four major side chains of the GAX oligosaccharides in the Coculture Broths.

Orange stars: d-Xylopyranose, green stars: l-Arabinofuranose, yellow circles: l-Galactopyranose, blue and white diamonds: d-glucuronic acid. The structures were determined using NMR spectroscopy and MALDI-TOF MS spectrometry. Details of the structural characterization are shown in Supplementary Figs. 1 and 3 and Supplementary Tables 1, 2, and 3.

Fig. 2. Activity of the purified enzymes against the Coculture Broth neutral oligosaccharides.

1D ¹H NMR analysis of products generated by individually incubating the purified enzymes with a mixture of neutral oligosaccharides (DP 4-9) isolated from TS-Coculture Broth. Data correspond to 2 h incubation beyond which no further activity was detected. The signals labeled terminal (T) α-l-Araf in the α-Araf_1120, α-Araf_996, and β-Xylp_1710 spectra correspond to T-α-l-Araf attached to O-3 to the backbone xylose. The T-α-l-Araf-label in the α-Xylp_1211 spectrum indicated the signal for T-α-l-Araf attached to O-2. The xylose at the reducing end of the oligosaccharide are labeled red. A scheme of the enzymatic reaction for each enzyme is included on the right side of the figure.

Fig. 3. Activity of the α-xylosidase α-Xylp_1211 against the Coculture Broth acidic oligosaccharide.

The GAX acidic oligosaccharide was isolated from the TS-Coculture Broth and incubated with the α-xylosidase α-Xylp_1211 for 2 h. The oligosaccharide was analyzed by NMR before and after the incubation with the enzyme. The oligosaccharide contains a disaccharide α-d-Xylp (1,3)-α-l-Araf-(1,2)- (A) and a T-β-d-Xylp-(1,3)-residue (B) that are attached to the double substituted 4-linked β-d-Xylp in the backbone (AB). The disappearance of signal for terminal (T) α-d-Xylp in the 1D ¹H NMR spectrum of the oligosaccharide after the incubation, indicates that the enzyme effectively removed this residue from A. A diagram of the acidic oligosaccharide structure and the enzymatic reaction is included at the top of the figure.

Fig. 4. Expression constructs for glycosyl hydrolase expression in T. thermosaccharolyticum.

a Construct for single enzyme expression, b construct for two genes which paired α-Xylp_1211 with another enzyme, and c construct for expression of four genes together; three such constructs, as shown in Supplementary Table 11, were bulit. CBP promoter: strong promoter from C. thermocellum that drives the cbp (Clo1313_1954) gene; Kan: Kanamycin resistance marker; E. coli/T. sacch replicon: contains origins of replication for E. coli and T. thermosaccharolyticum/T. saccharolyticum.

Fig. 5. Growth of T. thermosaccharolyticum strain LL1703 on Monoculture Broth.

The growth curves are representative of two replicates. The final % utilization (n = 6) was 65 ± 2% for the enzyme-expressing strain, 51 ± 1% for the parent stain, and 76 ± 2% for the strain supplemented with enzymes. Red circles—LL1548 (parent strain); black squares—LL1703 (LL1548 expressing four enzymes from LL1355); blue triangles—LL1548 with added LL1703 CFE. Source data are provided as a Source Data file.