Jose Serate

Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

Characterization of Variants Altered at the N-terminal Proline, a Novel Heme-Axial Ligand in CooA, the CO-sensing Transcriptional Activator

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro2 of one subunit (Met1 is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the resulting CooA variants. While Pro2appears to be optimal for CooA function, it is not essential and a variety of studied variants at this position have substantial CO-sensing function. Surprisingly, even alterations that add a residue (where Pro2 is replaced by Met1-Tyr2, for example) accumulate heme-containing CooA with functional properties that are similar to those of wild-type CooA. Other nearby residues, such as Phe5 and Asn6 appear to be important for either the structural integrity or the function of CooA. These results are contrasted with those previously reported for alteration of the His77 ligand on the opposite side of the heme.

Mutagenesis and Functional Characterization of the Four Domains of GlnD, a Bifunctional Nitrogen Sensor Protein

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) protein; P(II) in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

Ligand Responses of Vfr, the Virulence Factor Regulator from Pseudomonas aeruginosa

Vfr, a transcription factor homologous to the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP), regulates many aspects of virulence in Pseudomonas aeruginosa. Vfr, like CRP, binds to cAMP and then recognizes its target DNA and activates transcription. Here we report that Vfr has important functional differences from CRP in terms of ligand sensing and response. First, Vfr has a significantly higher cAMP affinity than does CRP, which might explain the mysteriously unidirectional functional complementation between the two proteins (S. E. H. West et al., J. Bacteriol. 176:7532-7542, 1994). Second, Vfr is activated by both cAMP and cGMP, while CRP is specific to cAMP. Mutagenic analyses show that Thr133 (analogous to Ser128 of CRP) is the key residue for both of these distinct Vfr properties. On the other hand, substitutions that cause cAMP-independent activity in Vfr are similar to those seen in CRP, suggesting that a common cAMP activation mechanism is present. In the course of these analyses, we found a remarkable class of Vfr variants that have completely reversed the regulatory logic of the protein: they are active in DNA binding without cAMP and are strongly inhibited by cAMP. The physiological impact of Vfrs ligand sensing and response is discussed, as is a plausible basis for the fundamental change in protein allostery in the novel group of Vfr variants.

Controlling microbial contamination during hydrolysis of AFEX-pretreated corn stover and switchgrass: effects on hydrolysate composition, microbial response and fermentation.

BackgroundMicrobial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics during the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates.ResultsAutoclaving the pretreated feedstocks could eliminate the contamination for a variety of feedstocks, whereas the antibiotic gentamicin was unable to control contamination consistently during hydrolysis. Compared to the addition of gentamicin, autoclaving of biomass before hydrolysis had a minimal effect on mineral concentrations, and showed no significant effect on the two major sugars (glucose and xylose) found in these hydrolysates. However, autoclaving elevated the concentration of some furanic and phenolic compounds. Chemical genomics analyses using Saccharomyces cerevisiae strains indicated a high correlation between the AFEX-pretreated hydrolysates produced using these two methods within the same feedstock, indicating minimal differences between the autoclaving and antibiotic methods. Comparative fermentations with S. cerevisiae and Zymomonas mobilis also showed that autoclaving the AFEX-pretreated feedstocks had no significant effects on microbial performance in these hydrolysates.ConclusionsOur results showed that autoclaving the pretreated feedstocks offered advantages over the addition of antibiotics for hydrolysate production. The autoclaving method produced a more consistent quality of hydrolysate, and also showed negligible effects on microbial performance. Although the levels of some of the lignocellulose degradation inhibitors were elevated by autoclaving the feedstocks prior to enzymatic hydrolysis, no significant effects on cell growth, sugar utilization, or ethanol production were seen during bacterial or yeast fermentations in hydrolysates produced using the two different methods.

Directed Evolution of the Escherichia coli cAMP Receptor Protein at the cAMP Pocket

Background: Thr127 and Ser128 are important for the function of the Escherichia coli CRP (cAMP receptor protein). Results: Thr/Ser, Thr/Thr, and Thr/Asn pairs at the positions are optimal for CRP function. Conclusion: Evolutionarily conserved residue pairs at the positions provide high cAMP affinity while keeping CRP inactive without cAMP. Significance: There are multiple evolutionary strategies for cAMP sensing in CRP. The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr127 and Ser128, are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr127 and Ser128 residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr127 and Ser128 was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

Diverse lignocellulosic feedstocks can achieve high field‐scale ethanol yields while providing flexibility for the biorefinery and landscape‐level environmental benefits

Increasing the diversity of lignocellulosic feedstocks accepted by a regional biorefinery has the potential to improve the environmental footprint of the facility; harvest, storage, and transportation logistics; and biorefinery economics. However, feedstocks can vary widely in terms of their biomass yields and quality characteristics (chemical composition, moisture content, etc.). To investigate how the diversity of potential biofuel cropping systems and feedstock supply might affect process and field‐scale ethanol yields, we processed and experimentally quantified ethanol production from five different herbaceous feedstocks: two annuals (corn stover and energy sorghum) and three perennials (switchgrass, miscanthus, and mixed prairie). The feedstocks were pretreated using ammonia fiber expansion (AFEX), hydrolyzed at high solid loading (~17%–20% solids, depending on the feedstock), and fermented separately using microbes engineered to utilize xylose: yeast (Saccharomyces cerevisiaeY128) or bacteria (Zymomonas mobilis8b). The field‐scale ethanol yield from each feedstock was dependent on biomass quality and cropping system productivity; however, biomass yield had a greater influence on the ethanol yield for low‐productivity crops, while biomass quality was the main driver for ethanol yields from high‐yielding crops. The process ethanol yield showed similar variability across years and feedstocks. A low process yield for corn stover was determined to result from inhibition of xylose utilization by unusually elevated levels of hydroxycinnamates (p‐coumaric and ferulic acids) in the untreated biomass and their acid and amide derivatives in the resulting hydrolyzate. This finding highlights the need to better understand factors that influence process ethanol yield and biomass quality. Ultimately we provide evidence that most feedstocks fall within a similar range of process ethanol yield, particularly for the more resistant strain Z. mobilis8b. This supports the claim that the refinery can successfully diversify its feedstock supply, enabling many social and environmental benefits that can accrue due to landscape diversification.

Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction

The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP’s recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.