Daniel G. Olson

Recent progress in consolidated bioprocessing.

Consolidated bioprocessing, or CBP, the conversion of lignocellulose into desired products in one step without added enzymes, has been a subject of increased research effort in recent years. In this review, the economic motivation for CBP is addressed, advances and remaining obstacles for CBP organism development are reviewed, and we comment briefly on fundamental aspects. For CBP organism development beginning with microbes that have native ability to utilize insoluble components of cellulosic biomass, key recent advances include the development of genetic systems for several cellulolytic bacteria, engineering a thermophilic bacterium to produce ethanol at commercially attractive yields and titers, and engineering a cellulolytic microbe to produce butanol. For CBP organism development, beginning with microbes that do not have this ability and thus requiring heterologous expression of a saccharolytic enzyme system, high-yield conversion of model cellulosic substrates and heterologous expression of CBH1 and CBH2 in yeast at levels believed to be sufficient for an industrial process have recently been demonstrated. For both strategies, increased emphasis on realizing high performance under industrial conditions is needed. Continued exploration of the underlying fundamentals of microbial cellulose utilization is likely to be useful in order to guide the choice and development of CBP systems.

High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes

ABSTRACT This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.

Development of pyrF-Based Genetic System for Targeted Gene Deletion in Clostridium thermocellum and Creation of a pta Mutant

ABSTRACT We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Δpta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.

Deletion of the Cel48S cellulase from Clostridium thermocellum

Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose with the aid of a multienzyme cellulosome complex. Creation of knockout mutants for Cel48S (also known as CelS, SS, and S8), the most abundant cellulosome subunit, was undertaken to gain insight into its role in enzymatic and microbial cellulose solubilization. Cultures of the Cel48S deletion mutant (S mutant) were able to completely solubilize 10 g/L crystalline cellulose. The cellulose hydrolysis rate of the S mutant strain was 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. The cellulosome produced by the S mutant strain was purified by affinity digestion, characterized enzymatically, and found to have a 35% lower specific activity on Avicel. The composition of the purified cellulosome was analyzed by tandem mass spectrometry with APEX quantification and no significant changes in abundance were observed in any of the major (>1% of cellulosomal protein) enzymatic subunits. Although most cellulolytic bacteria have one family 48 cellulase, C. thermocellum has two, Cel48S and Cel48Y. Cellulose solubilization by a Cel48S and Cel48Y double knockout was essentially the same as that of the Cel48S single knockout. Our results indicate that solubilization of crystalline cellulose by C. thermocellum can proceed to completion without expression of a family 48 cellulase.

Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model

UNLABELLED The airways of patients with cystic fibrosis are colonized with diverse bacterial communities that change dynamically during pediatric years and early adulthood. Staphylococcus aureus is the most prevalent pathogen during early childhood, but during late teens and early adulthood, a shift in microbial composition occurs leading to Pseudomonas aeruginosa community predominance in ∼50% of adults. We developed a robust dual-bacterial in vitro coculture system of P. aeruginosa and S. aureus on monolayers of human bronchial epithelial cells homozygous for the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation to better model the mechanisms of this interaction. We show that P. aeruginosa drives the S. aureus expression profile from that of aerobic respiration to fermentation. This shift is dependent on the production of both 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and siderophores by P. aeruginosa. Furthermore, S. aureus-produced lactate is a carbon source that P. aeruginosa preferentially consumes over medium-supplied glucose. We find that initially S. aureus and P. aeruginosa coexist; however, over extended coculture P. aeruginosa reduces S. aureus viability, also in an HQNO- and P. aeruginosa siderophore-dependent manner. Interestingly, S. aureus small-colony-variant (SCV) genetic mutant strains, which have defects in their electron transport chain, experience reduced killing by P. aeruginosa compared to their wild-type parent strains; thus, SCVs may provide a mechanism for persistence of S. aureus in the presence of P. aeruginosa. We propose that the mechanism of P. aeruginosa-mediated killing of S. aureus is multifactorial, requiring HQNO and P. aeruginosa siderophores as well as additional genetic, environmental, and nutritional factors. IMPORTANCE In individuals with cystic fibrosis, Staphylococcus aureus is the primary respiratory pathogen during childhood. During adulthood, Pseudomonas aeruginosa predominates and correlates with worse patient outcome. The mechanism(s) by which P. aeruginosa outcompetes or kills S. aureus is not well understood. We describe an in vitro dual-bacterial species coculture system on cystic fibrosis-derived airway cells, which models interactions relevant to patients with cystic fibrosis. Further, we show that molecules produced by P. aeruginosa additively induce a transition of S. aureus metabolism from aerobic respiration to fermentation and eventually lead to loss of S. aureus viability. Elucidating the molecular mechanisms of P. aeruginosa community predominance can provide new therapeutic targets and approaches to impede this microbial community transition and subsequent patient worsening.

Transformation of Clostridium Thermocellum by Electroporation

In this work, we provide detailed instructions for transformation of Clostridium thermocellum by electroporation. In addition, we describe two schemes for genetic modification: allelic replacement-where the gene of interest is replaced by an antibiotic marker and markerless gene deletion-where the gene of interest is removed and the selective markers are recycled. The markerless gene deletion technique can also be used for insertion of genes onto the C. thermocellum chromosome.

Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum

BackgroundThe native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol.ResultsH2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ∆hydG∆ech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ∆hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, ∆hydG and ∆hydG∆ech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum.ConclusionsThe dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

BackgroundIndustrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies.ResultsWe report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+dcm+E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude.ConclusionsE. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities

The multi–length scale nature of its glycoside hydrolase system explains the remarkable ability demonstrated by Clostridium thermocellum. Clostridium thermocellum is the most efficient microorganism for solubilizing lignocellulosic biomass known to date. Its high cellulose digestion capability is attributed to efficient cellulases consisting of both a free-enzyme system and a tethered cellulosomal system wherein carbohydrate active enzymes (CAZymes) are organized by primary and secondary scaffoldin proteins to generate large protein complexes attached to the bacterial cell wall. This study demonstrates that C. thermocellum also uses a type of cellulosomal system not bound to the bacterial cell wall, called the “cell-free” cellulosomal system. The cell-free cellulosome complex can be seen as a “long range cellulosome” because it can diffuse away from the cell and degrade polysaccharide substrates remotely from the bacterial cell. The contribution of these two types of cellulosomal systems in C. thermocellum was elucidated by characterization of mutants with different combinations of scaffoldin gene deletions. The primary scaffoldin, CipA, was found to play the most important role in cellulose degradation by C. thermocellum, whereas the secondary scaffoldins have less important roles. Additionally, the distinct and efficient mode of action of the C. thermocellum exoproteome, wherein the cellulosomes splay or divide biomass particles, changes when either the primary or secondary scaffolds are removed, showing that the intact wild-type cellulosomal system is necessary for this essential mode of action. This new transcriptional and proteomic evidence shows that a functional primary scaffoldin plays a more important role compared to secondary scaffoldins in the proper regulation of CAZyme genes, cellodextrin transport, and other cellular functions.

Ethanol production by engineered thermophiles

We compare a number of different strategies that have been pursued to engineer thermophilic microorganisms for increased ethanol production. Ethanol production from pyruvate can proceed via one of four pathways, which are named by the key pyruvate dissimilating enzyme: pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH), pyruvate formate lyase (PFL), and pyruvate ferredoxin oxidoreductase (PFOR). For each of these pathways except PFL, we see examples where ethanol production has been engineered with a yield of >90% of the theoretical maximum. In each of these cases, this engineering was achieved mainly by modulating expression of native genes. We have not found an example where a thermophilic ethanol production pathway has been transferred to a non-ethanol-producing organism to produce ethanol at high yield. A key reason for the lack of transferability of ethanol production pathways is the current lack of understanding of the enzymes involved.