Eleftherios T. Papoutsakis

A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: From biofuels and chemicals, to biocatalysis and bioremediation

Metabolites, substrates and substrate impurities may be toxic to cells by damaging biological molecules, organelles, membranes or disrupting biological processes. Chemical stress is routinely encountered in bioprocessing to produce chemicals or fuels from renewable substrates, in whole-cell biocatalysis and bioremediation. Cells respond, adapt and may develop tolerance to chemicals by mechanisms only partially explored, especially for multiple simultaneous stresses. More is known about how cells respond to chemicals, but less about how to develop tolerant strains. Aiming to stimulate new metabolic engineering and synthetic-biology approaches for tolerant-strain development, this review takes a holistic, comparative and modular approach in bringing together the large literature on genes, programs, mechanisms, processes and molecules involved in chemical stress or imparting tolerance. These include stress proteins and transcription factors, efflux pumps, altered membrane composition, stress-adapted energy metabolism, chemical detoxification, and accumulation of small-molecule chaperons and compatible solutes. The modular organization (by chemicals, mechanism, organism, and methods used) imparts flexibility in exploring this complex literature, while comparative analyses point to hidden commonalities, such as an oxidative stress response underlying some solvent and carboxylic-acid stress. Successes involving one or a few genes, as well as global genomic approaches are reviewed with an eye to future developments that would engage novel genomic and systems-biology tools to create altered or semi-synthetic strains with superior tolerance characteristics for bioprocessing.

Overexpression of groESL in Clostridium acetobutylicum Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program

ABSTRACT DNA array and Western analyses were used to examine the effects of groESL overexpression and host-plasmid interactions on solvent production in Clostridium acetobutylicum ATCC 824. Strain 824(pGROE1) was created to overexpress the groESL operon genes from a clostridial thiolase promoter. The growth of 824(pGROE1) was inhibited up to 85% less by a butanol challenge than that of the control strain, 824(pSOS95del). Overexpression of groESL resulted in increased final solvent titers 40% and 33% higher than those of the wild type and plasmid control strains, respectively. Active metabolism lasted two and one half times longer in 824(pGROE1) than in the wild type. Transcriptional analysis of 824(pGROE1) revealed increased expression of motility and chemotaxis genes and a decrease in the expression of the other major stress response genes. Decreased expression of the dnaKJ operon upon overexpression of groESL suggests that groESL functions as a modulator of the CIRCE regulon, which is shown here to include the hsp90 gene. Analysis of the plasmid control strain 824(pSOS95del) revealed complex host-plasmid interactions relative to the wild-type strain, resulting in prolonged biphasic growth and metabolism. Decreased expression of four DNA gyrases resulted in differential expression of many key primary metabolism genes. The ftsA and ftsZ genes were expressed at higher levels in 824(pSOS95del), revealing an altered cell division and sporulation pattern. Both transcriptional and Western analyses revealed elevated stress protein expression in the plasmid-carrying strain.

Engineering solventogenic clostridia

Solventogenic clostridia are strictly anaerobic, endospore forming bacteria that produce a large array of primary metabolites, like butanol, by anaerobically degrading simple and complex carbohydrates, including cellulose and hemicellulose. Two genomes have been sequenced and some genetic tools have been developed, but more are now urgently needed. Genomic tools for designing, and assessing the impact of, genetic modifications are well developed. Early efforts to metabolically engineer these organisms suggest that they are promising organisms for biorefinery applications. Pathway engineering efforts have resulted in interesting strains, but global engineering of their transcriptional machinery has produced better outcomes. Future efforts are expected to undertake the development of complex multigenic phenotypes, such as aerotolerance, solvent tolerance, high-cell density fermentations, abolished sporulation without impacting product formation, and genetic stability for continuous bioprocessing.

A comparative genomic view of clostridial sporulation and physiology.

Clostridia are anaerobic, endospore-forming prokaryotes that include strains of importance to human and animal health and physiology, cellulose degradation, solvent production and bioremediation. Their differentiation and related developmental programmes are not well understood at the molecular level. Recent genome sequencing and transcriptional-profiling studies have offered a glimpse of their inner workings and indicate that a better understanding of the orchestration of the molecular events that underlie their unique physiology, capabilities and diversity will pay major dividends.

Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications.

Clostridia are anaerobic Firmicutes producing a large array of metabolites by utilizing simple and complex carbohydrates, such as cellulose, as well as CO2/H2 or CO. Their exceptional substrate diversity is enhanced by their ability to produce a broad spectrum of chemicals that can be used as precursors to or directly as biofuels and industrial chemicals. Genetic and genomic tools are under intense development, and recent efforts to metabolically engineer clostridia demonstrate their potential for biofuel and biorefinery applications. Pathway engineering to combine established substrate-utilization programs, such as for cellulose, CO2/H2 or CO, with desirable metabolic programs could lead to modular design of strains suitable for many applications. Engineering complex phenotypes--aerotolerance, abolished sporulation, and tolerance to toxic chemicals--could lead to superior bioprocessing strains.

Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824

Integrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in Clostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

Fluid-mechanical damage of animal cells in bioreactors

The fluid-mechanical and some biological aspects of damage to animal cells in bioreactors due to agitation and/or aeration are attracting renewed attention. In microcarrier bioreactors, cell damage is due to forces generated by the interaction of microcarrier beads with each other and also with small turbulent eddies. For freely suspended cells grown in mixed bioreactors, cell damage is most frequently due to bubble breakup or fast-draining liquid films around rearranging gas-liquid interfaces.

Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

The effects of challenges with low (0.25%, vol/vol) and high (0.75%) concentrations of butanol on the growth, glucose metabolism, product formation, and transcriptional program of the solvent-tolerant Clostridium acetobutylicum strain 824(pGROE1) and the plasmid control strain 824(pSOS95del) were used to study solvent tolerance and stress response. Strain 824(pGROE1) was generated by groESL overexpression. The growth of 824(pGROE1) was less inhibited than that of 824(pSOS95del), and 824(pGROE1) was able to metabolize glucose over the entire course of the culture (60 h postchallenge) while glucose metabolism in 824(pSOS95del) lasted 24 h. A comparison of their respective DNA array-based transcriptional profiles identified genes with similar expression patterns (these genes are likely to be part of a general butanol stress response) and genes with opposite expression patterns (these genes are likely to be associated with increased tolerance to butanol). Both strains exhibited a butanol dose-dependent increase in expression of all major stress protein genes, including groES, dnaKJ, hsp18, and hsp90; all major solvent formation genes, including aad, ctfA and -B, adc, and bdhA and -B (an unexpected and counterintuitive finding); the butyrate formation genes (ptb and buk); the butyryl coenzyme A biosynthesis operon genes; fructose bisphosphate aldolase; and a gene with homology to Bacillus subtilis kinA. A dose-dependent decrease in expression was observed for the genes of the major fatty acid synthesis operon (also an unexpected and counterintuitive finding), several glycolytic genes, and a few sporulation genes. Genes with opposite expression kinetics included rlpA, artP, and a gene encoding a hemin permease. Taken together, these data suggest that stress, even when it derives from the solvent product itself, triggers the induction of the solvent formation genes.

Northern, Morphological, and Fermentation Analysis of spo0A Inactivation and Overexpression in Clostridium acetobutylicum ATCC 824

The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration--a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B. subtilis. Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.

Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: Need for new phenomenological models for solventogenesis and butanol inhibition?

Two metabolic engineering tools, namely gene inactivation and gene overexpression, were employed to examine the effects of two genetic modifications on the fermentation characteristics of Clostridium acetobutylicum. Inactivation of the butyrate kinase gene (buk) was examined using strain PJC4BK, while the combined effect of buk inactivation and overexpression of the aad gene-encoding the alcohol aldehyde dehydrogense (AAD) used in butanol formation-was examined using strain PJC4BK(pTAAD). The two strains were characterized in controlled pH > or = 5.0 fermentations, and by a recently enhanced method of metabolic flux analysis. Strain PJC4BK was previously genetically characterized, and fermentation experiments at pH > or = 5.5 demonstrated good, but not exceptional, solvent-production capabilities. Here, we show that this strain is a solvent superproducer in pH > or = 5.0 fermentations producing 225 mM (16.7 g/L) of butanol, 76 mM of acetone (4.4 g/L), and 57 mM (2.6 g/L) of ethanol. Strain PJC4BK(pTAAD) produced similar amounts of butanol and acetone but 98 mM (4.5 g/L) of ethanol. Both strains overcame the 180 mM (13 g/L) butanol toxicity limit, without any selection for butanol tolerance. Work with strain PJC4BK(pTAAD) is the first reported use of dual antibiotic selection in C. acetobutylicum. One antibiotic was used for selection of strain PJC4BK while the second antibiotic selected for the pTAAD presence. Overexpression of aad from pTAAD resulted in increased ethanol production but did not increase butanol titers, thus indicating that AAD did not limit butanol production under these fermentation conditions. Metabolic flux analysis showed a decrease in butyrate formation fluxes by up to 75% and an increase in acetate formation fluxes of up to 100% during early growth. The mean specific butanol and ethanol formation fluxes increased significantly in these recombinant strains, up to 300% and 400%, respectively. Onset of solvent production occurred during the exponential-growth phase when the culture optical density was very low and when total and undissociated butyric acid levels were <1 mM. Butyrate levels were low throughout all fermentations, never exceeding 20 mM. Thus, threshold butyrate concentrations are not necessary for solvent production in these stains, suggesting the need for a new phenomenological model to explain solvent formation.