Shengde Zhou

Bio: Shengde Zhou is an academic researcher from Northern Illinois University. The author has contributed to research in topics: Fermentation & Escherichia coli. The author has an hindex of 26, co-authored 45 publications receiving 2681 citations. Previous affiliations of Shengde Zhou include University of Florida & Hubei University of Technology.

Enteric bacterial catalysts for fuel ethanol production

Abstract: The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.

Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110.

Abstract: The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of l-lactic acid to d-lactic acid. Although the largest demand is for the l enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of d-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. d-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this d-lactic acid was ∼98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (ackA). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of d-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.

Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: Homoacetate production

Abstract: Microbial processes for commodity chemicals have focused on reduced products and anaerobic conditions where substrate loss to cell mass and CO2 are minimal and product yields are high. To facilitate expansion into more oxidized chemicals, Escherichia coli W3110 was genetically engineered for acetate production by using an approach that combines attributes of fermentative and oxidative metabolism (rapid growth, external electron acceptor) into a single biocatalyst. The resulting strain (TC36) converted 333 mM glucose into 572 mM acetate, a product of equivalent oxidation state, in 18 h. With excess glucose, a maximum of 878 mM acetate was produced. Strain TC36 was constructed by sequentially assembling deletions that inactivated oxidative phosphorylation (ΔatpFH), disrupted the cyclic function of the tricarboxylic acid pathway (ΔsucA), and eliminated native fermentation pathways (ΔfocA-pflB ΔfrdBC ΔldhA ΔadhE). These mutations minimized the loss of substrate carbon and the oxygen requirement for redox balance. Although TC36 produces only four ATPs per glucose, this strain grows well in mineral salts medium and has no auxotrophic requirement. Glycolytic flux in TC36 (0.3 μmol⋅min−1⋅mg−1 protein) was twice that of the parent. Higher flux was attributed to a deletion of membrane-coupling subunits in (F1F0)H+-ATP synthase that inactivated ATP synthesis while retaining cytoplasmic F1-ATPase activity. The effectiveness of this deletion in stimulating flux provides further evidence for the importance of ATP supply and demand in the regulation of central metabolism. Derivatives of TC36 may prove useful for the commercial production of a variety of commodity chemicals.

Re-engineering Escherichia coli for ethanol production.

Abstract: A lactate producing derivative of Escherichia coli KO11, strain SZ110, was re-engineered for ethanol production by deleting genes encoding all fermentative routes for NADH and randomly inserting a promoterless mini-Tn5 cassette (transpososome) containing the complete Zymomonas mobilis ethanol pathway (pdc, adhA, and adhB) into the chromosome. By selecting for fermentative growth in mineral salts medium containing xylose, a highly productive strain was isolated in which the ethanol cassette had been integrated behind the rrlE promoter, designated strain LY160(KO11, Deltafrd::celY(Ec) DeltaadhE DeltaldhA, DeltaackA lacA::casAB(Ko) rrlE::(pdc( Zm)-adhA(Zm)-adhB(Zm)-FRT-rrlE)pflB(+)). This strain fermented 9% (w/v) xylose to 4% (w/v) ethanol in 48 h in mineral salts medium, nearly equal to the performance of KO11 with Luria broth.

Microbial cellulose utilization: fundamentals and biotechnology.

Abstract: Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels

Abstract: Global energy and environmental problems have stimulated increased efforts towards synthesizing biofuels from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. In addition, branched-chain alcohols have higher octane numbers compared with their straight-chain counterparts. However, these alcohols cannot be synthesized economically using native organisms. Here we present a metabolic engineering approach using Escherichia coli to produce higher alcohols including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from glucose, a renewable carbon source. This strategy uses the host's highly active amino acid biosynthetic pathway and diverts its 2-keto acid intermediates for alcohol synthesis. In particular, we have achieved high-yield, high-specificity production of isobutanol from glucose. The strategy enables the exploration of biofuels beyond those naturally accumulated to high quantities in microbial fermentation.

Hemicellulose bioconversion

Abstract: Various agricultural residues, such as corn fiber, corn stover, wheat straw, rice straw, and sugarcane bagasse, contain about 20–40% hemicellulose, the second most abundant polysaccharide in nature. The conversion of hemicellulose to fuels and chemicals is problematic. In this paper, various pretreatment options as well as enzymatic saccharification of lignocellulosic biomass to fermentable sugars is reviewed. Our research dealing with the pretreatment and enzymatic saccharification of corn fiber and development of novel and improved enzymes such as endo-xylanase, β-xylosidase, and α-l-arabinofuranosidase for hemicellulose bioconversion is described. The barriers, progress, and prospects of developing an environmentally benign bioprocess for large-scale conversion of hemicellulose to fuel ethanol, xylitol, 2,3-butanediol, and other value-added fermentation products are highlighted.