Neil E. Welker

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.

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.

Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript. Western analysis showed that all three strains exhibit low levels of AADC compared to the plasmid control [ATCC 824(pSOS95del)]. By using computational algorithms, the three different asRNAs directed toward AADC, along with previously reported clostridial asRNAs, were examined for structural features (free nucleotides and components). When the normalized metrics of these structural features were plotted against percent downregulation, only the component/nucleotide ratio correlated well with in vivo asRNA effectiveness. Despite the significant downregulation of AADC in these strains, there were no concomitant effects on acetone formation. These findings suggest that AADC does not limit acetone formation and, thus, we targeted next the CoAT. Using the component/nucleotide ratio as a selection parameter, we developed three strains [ATCC 824 (pCTFA2AS), 824(pCTFB1AS), and 824(pCOAT11AS)] which express asRNAs to downregulate either or both of the CoAT subunits. Compared to the plasmid control strain, these strains produced substantially low levels of acetone and butanol and Western blot analyses showed significantly low levels of both CoAT subunits. These results show that CoAT is the rate-limiting enzyme in acetone formation and strengthen the hypothesis that the component/nucleotide ratio is a predictive indicator of asRNA effectiveness.

Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production. Journal of Industrial Microbiology & Biotechnology (2001) 27, 322–328.

Wind River Conference on Prokaryotic Biology—2002

The Wind River Conference, which was begun as the Transformation Meeting in 1957, was renamed the Wind River Conference on Genetic Exchange and ultimately became the Wind River Conference on Prokaryotic Biology to expand the range of areas of discussion and presentation ([3][1]). Initially, the