Escherichia coli binary culture engineered for direct fermentation of hemicellulose to a biofuel

Abstract

Metabolic engineering has created several Escherichia coli biocatalysts for production of biofuels and other useful molecules. However, the inability of these biocatalysts to directly use polymeric substrates necessitates costly pretreatment and enzymatic hydrolysis prior to fermentation. Consolidated bioprocessing has the potential to simplify the process by combining enzyme production, hydrolysis, and fermentation into a single step but requires a fermenting organism to multitask by producing both necessary enzymes and target molecules. We demonstrate here a binary strategy for consolidated bioprocessing of xylan, a complex substrate requiring six hemicellulases for complete hydrolysis. An integrated modular approach was used to design the two strains to function cooperatively in the process of transforming xylan into ethanol. The first strain was engineered to coexpress two hemicellulases. Recombinant enzymes were secreted to the growth medium by a method of lpp deletion with over 90% efficiency. Secreted enzymes hydrolyzed xylan into xylooligosaccharides, which were taken in by the second strain, designed to use the xylooligosaccharides for ethanol production. Cocultivation of the two strains converted xylan hemicellulose to ethanol with a yield about 55% of the theoretical value. Inclusion of other three hemicellulases improved the ethanol yield to 70%. Analysis of the culture broth showed that xylooligosaccharides with four or more xylose units were not utilized, suggesting that improving the use of higher xyloogligomers should be the focus in future efforts. This is the first demonstration of an engineered binary culture for consolidated bioprocessing of xylan. The modular design should allow the strategy to be adopted for a broad range of biofuel and biorefinery products.

FIG. 1.

A xylan structure and a hemicellulase system for its hydrolysis. Abbreviations: Xyl, β-d-xylose; Abf, α-l-arabinofuranose; Fer, ferulic acid; Act, acetyl group; mGu, α-O-methyl-d-glucuronic acid.

FIG. 2.

Maps of recombinant plasmids pCRAXEXYL (A), pCRAXEXYL(Km⁻) (B), pBBKXYN (C), and pTAGU (D). Abbreviations: P_Lac, lac promoter; P_T5, T5 promoter; P_Tac, tac promoter; malE^s, signal peptide sequence of MalE protein; tat^s, twin arginine TorA signal peptide sequence of trimethylamine N-oxide reductase; axeA, acetylxylan esterase axe gene from Streptomyces violaceoruber; xyl11A, xylanase gene from Bacillus halodurans C-125 (BH0899); KxynB, β-xylosidase gene from Klebsiella pneumonia ATCC 700721; KxynT, xyloside permease gene from Klebsiella pneumoniae ATCC 700721; agu67A, α-glucuronidase gene from Cellvibrio japonicas; ori, ColE1 replication origin; Rep, replication of origin for pBBR122; ApR, ampicillin resistance gene; KmR, kanamycin resistance gene.

FIG. 3.

Binary system designed for conversion of xylan to ethanol. The binary system consists of two E. coli strains, E609Y and KO11, with both chromosomal and plasmid-borne genetic modifications, as indicated in the illustration. Abbreviations: P_Lac, lac promoter; P_T5, T5 promoter; axeA, acetylxylan esterase gene from Streptomyces violaceoruber; xyl11A, xylanase gene from Bacillus halodurans C-125 (BH0899); KxynB, β-xylosidase gene from Klebsiella pneumoniae ATCC 700721; KxynT, xyloside permease gene from Klebsiella pneumoniae ATCC 700721; lpp⁻, murein lipoprotein gene deletion mutant of E. coli; pdc, pyruvate decarboxylase gene from Zymomonas mobilis ZM4; adh, alcohol dehydrogenase gene from Zymomonas mobilis ZM4; ori, ColE1 replication origin; Rep, origin of replication for pBBR122.

FIG. 4.

SDS-PAGE of whole-protein extract of KO11 expressing β-xylosidase (KXynB).

FIG. 5.

Colonies from binary culture on Congo red-xylan plates at different times of cocultivation.

FIG. 6.

Effect of postinduction time on ethanol concentration.

FIG. 7.

(A) Time profiles of cell density, reducing sugar, and ethanol concentration during cocultivation of the engineered strains with exogenous α-arabinofuranosidase and ferulic acid esterase. Symbols: ▪, reducing sugar; ⧫, cell growth; ▴, ethanol. (B) HPAEC spectra of broth samples of binary culture at 0 and 24 h of cocultivation.