3.4.1 "Traditional" Microbial Ethanologens

Given the present indeterminacy regarding “best” choices for producing organism and biomass substrate, it is timely to summarize recent advances in the metabolic and genetic sciences relevant to yeast and bacterial ethanologens, and from them to project forward key areas for their application to industrial ethanol production after 2007. Four themes can readily be identified:

1. A better understanding of stress responses

2. Relating transport phenomena to sugar utilization and ethanol productivity

3. Defining the roles of particular genes in the metabolism of ethanologens

4. Applying whole-genome methodologies and in silico simulations to opti­mize the balance of carbon flows along synergistic or competing pathways

Yeast ethanologens show dose-dependent inhibition by furfural and HMF and are generally more sensitive to furfural; a S. cerevisiae strain converted HMF to 2,5-bis-HMF, a previously postulated alcohol formed from the parent aldehyde, and this suggests that an enzymic pathway for detoxifying these components of lignocel- lulosic hydrolysates should be attainable by gene cloning and/or mutation to improve the activities and substrate preferences of the enzymes.244 That such a reduction reaction as that to form 2,5-bis-HMF might utilize NADPH as cofactor was shown by screening a gene disruption library from S. cerevisiae: one type of mutation was in zwfl, the gene encoding glucose 6-phosphate dehydrogenase, the NADPH-generating entry point to the oxidative pentose pathway; overexpression of zwfl allowed growth in furfural concentrations normally toxic, whereas similar effects with non-redox- associated gene products could be explained as a result of inhibiting the overall activ­ity of the pathway.245 The ADH6 gene of S. cerevisiae encodes a reductase enzyme that reduces HMF and NADPH supports a specific activity of the enzyme some 150­fold higher than with NADH; yeast strains overexpressing this gene had a higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures.246

A key trait for biofuels research is to enable growth in the presence of very high concentrations of both glucose and ethanol, the former in the batch medium (discussed in chapter 4, section 4.3), the latter accumulating as the fermentation proceeds. In yeasts, analysis of the effects of ethanol concentration and tempera­ture point to nonspecific actions on the cell membranes as leading to the variety of cellular responses, including reduced fermentation rate and viability.247 Adapting cells to ethanol causes changes in the cellular membrane composition, in particu­lar the degree of unsaturation of the fatty acids and the amount of sterols present, both indicative of alterations in the fluidity characteristics of the lipid-based mem­branes.248 The low tolerance of Pichia stipitis to ethanol can be correlated with a disruption of protein transport across the cell membrane, an energy-requiring pro­cess involving a membrane-localized ATPase; the system is activated by glucose, but ethanol uncouples ATP hydrolysis from protein transport, thus wasting energy for no purpose, and structural alteration of the ATPase to affect its sensitivity to ethanol could be a quick-win option for yeast ethanologens in general.249

In Z. mobilis, there is evidence for a glucose-sensing system to confer (or increase) the cells’ tolerance to the osmotic stress imposed by high sugar concentrations; the exact functions of the products of the four genes simultaneously transcribed in a glu­cose-regulated operon are unknown, but manipulating this genetic locus would aid adaptation to high concentrations (>100 g/l) of glucose in industrial media.250 The announcement of the complete sequencing of the genome of Z. mobilis by scientists in Seoul, South Korea, in 2005 provided rationalizations for features of the idio­syncratic carbohydrate metabolism in the absence of three key genes for the EMP/ tricarboxylic acid cycle paradigm, that is, 6-phosphofructokinase, 2-oxoglutarate dehydrogenase, and malate dehydrogenase.251 The nonoxidative pentose phosphate pathway is mostly missing, although the genes are present for the biosynthesis of phosphorylated ribose and thence histidine and nucleotides for both DNA and RNA (figure 3.12). The 2-Mbp circular chromosome encodes for 1,998 predicted func­tional genes; of these, nearly 20% showed no similarities to known genes, suggesting a high probability of coding sequences of industrial significance for ethanologens. In comparison to a strain of Z. mobilis with lower tolerance to ethanol and rates of ethanol production, glucose uptake, and specific growth rate, strain ZM4 contains 54 additional genes, including four transport proteins and two oxidoreductases, all potentially mediating the higher ethanol productivity of the strain. Moreover, two genes coding for capsular carbohydrate synthesis may be involved in generating an altered morphology more resistant to osmotic stress. Intriguingly, 25 of the “new” genes showed similarities to bacteriophage genes, indicating a horizontal transfer of genetic material via phages.251

Fructose

6-Phosphogluconate

2-Oxo-3-deoxy-6-phosphogluconate

Glyceraldehyde 3-phosphate

Ribulose 5-phosphate

1,3-Diphosphoglycerate

2-Phosphoglycerate

5-Phosphoribosyl pyrophosphate

Phosphoc^o/pyruvate

Oxaloacetate

Succinate

FIGURE 3.12 Fragmentary carbohydrate metabolism and interconversions in Z. mobilis as confirmed by whole-genome sequencing. (From Seo et al.251)

Analysis of mutants of the yeast Yarrowia lipolytica hypersensitive to acetic acid and ethanol identified a single gene that could direct multiple phenotypic changes, including colony morphology, the morphology of intracellular membranes, and the appearance of vacuoles and mitochondria, and induce early death on glucose even with­out the presence of acetate or ethanol; this GPR1 gene may be a target for manipulation
and mutation to increase solute tolerance.252 A more direct approach in S. cerevisiae involved multiple mutagenesis of a transcription factor and selection of dominant muta­tions that conferred increased ethanol tolerance and more efficient glucose conversion to ethanol: 20% for growth in the presence of an initial glucose concentration of 100 g/l, 69% for space-time yield (grams of ethanol per liter per hour), 41% for specific pro­ductivity, and 15% for ethanol yield from glucose.253 It is likely that the expression of an ensemble of hundreds of genes was increased by the mutations; of the 12 most over­expressed genes, deletions mutations abolished the increased glucose tolerance in all but one gene, and selective overexpression of each of the top three up-regulated genes could replicate the tolerance of the best mutant. Because this work was, by necessity, carried out on a laboratory haploid strain, it is possible that similar changes could have occurred gradually during many generations in industrial strains. The effect on gene expression was, however, greatly diminished at 120 g/l of glucose.

Comparable analyses of gene expression and function are now being introduced into commercial potable alcohol research. A common driver between breweries and industrial bioethanol plants is (or will be) the use of concentrated, osmotically stressful fermentation media. In breweries, the capability to use high-gravity brewing in which the wort is more concentrated than in traditional practice saves energy, time, and space; the ethanol-containing spent wort can be diluted either with lower alcohol-content wort or with water. Variants of brewer’s yeast (S. pastorianus) were obtained subjecting UV- mutagenized cells to consecutive rounds of fermentation in very-high-gravity wort; variants showing faster fermentation times and/or more complete sugar utilization were identified for investigations of gene expression with microarray technologies.254 Of the 13 genes either overexpressed or underexpressed in comparison with a control strain, a hexokinase gene was potentially significant as signaling a reduced carbon catabolite repression of maltose and maltotriose uptake; more speculatively, two ampli­fied genes in amino acid biosynthesis pathways could point to increased requirements for amino acid production for growth in the highly concentrated medium. Although deletion mutants are usually difficult to isolate with polyploid industrial strains, the dose-variable amplification of target genes is more feasible.

Strains of S. cerevisiae genetically engineered for xylose utilization depend, unlike naturally xylose-consuming yeasts, on xylose uptake via the endogenous hexose transporters; with a high XR activity, xylose uptake by this ad hoc arrange­ment limits the pentose utilization rate at low xylose concentrations, suggesting that expression of a yeast xylose/pentose transporter in S. cerevisiae would be beneficial for the kinetics and extent of xylose consumption from hemicellulose hydrolysates.255 A high-affinity xylose transporter is present in the cell membranes of the efficient xylose fermenting yeast Candida succiphila when grown on xylose and in Kluyvero — myces marxianus when grown on glucose under fully aerobic conditions.256

In ethanologenic bacteria, there are also grounds for postulating that radically chang­ing the mechanism of xylose uptake would improve both anaerobic growth and xylose utilization. Mutants of E. coli lacking PFL or acetate kinase activity fail to grow anaero­bically on xylose, that is, the cells are energy-limited without the means to generate ATP from acetyl phosphate; in contrast, the mutants can grow anaerobically on arabinose — and the difference resides in the sugar transport mechanisms: energy-expending active transport for xylose but an energy-conserving arabinose.257 Reconfiguring xylose uptake

is, therefore, an attractive option for removing energetic constraints on xylose metabo­lism. In yeast also, devising means of conserving energy during xylose metabolism would reduce the bioenergetic problems posed by xylose catabolism — gene expres­sion profiles during xylose consumption resemble those of the starvation response, and xylose appears to induce metabolic behavioral responses that mix features of those elic­ited by genuinely fermentable and respirative carbon sources.258

Looking beyond purely monomeric sugar substrates, the soil bacterium Geobacil­lus stearothermophilus is highly efficient at degrading hemicelluloses, possessing a 30- gene array for this purpose, organized into nine transcriptional units; in the presence of a xylan, endo-1,4-P-xylanase is secreted, and the resulting xylooligosaccharides enter the cell by a specialized transport system, now known to be a three-gene unit encod­ing an ATP-binding cassette transport system, whose transcription is repressed by xylose but activated by a response regulator.259 This regulation allows the cell to rapidly amplify the expression of the transport system when substrates are available. Inside the cell, the oligosaccharides are hydrolyzed to monomeric sugars by the actions of a fam­ily of xylanases, P-xylosidase, xylan acetylesterases, arabinofuranosidases, and gluc­uronidases. Such a complete biochemical toolkit for hemicellulose degradation should be considered for heterologous expression in bacterial bioethanol producers.

Control of the gene encoding the major PDC in wild-type S. cerevisiae was shown by analysis of gene transcription to be the result of ethanol repression rather than glucose induction; an ethanol-repression ERA sequence was identified in the yeast DNA; the Era protein binds to the PDC1 promoter sequence under all growth conditions, and an autoregulatory (A) factor stimulates PDC1 transcription but also binds to the pdc1 enzyme — the more enzyme is formed, the more A factor is bound, thus making the transcription of the FDC1 gene self-regulating.260 This may be a useful target for strain improvement; industrial strains may, however, have already been “blindly” selected at this locus.

Focusing on single-gene traits, a research group at Tianjin University (China) has recently demonstrated three approaches to reduce glycerol, acetate, and pyruvate accumulation during ethanol production by S. cerevisiae:

1. Simply deleting the gene encoding glycerol formation from glycolysis, that is, glycerol 3-phosphate dehydrogenase reduced the growth rate compared with the wild type but reduced acetate and pyruvate accumulation. Simul­taneously overexpressing the GLTl gene for glutamate synthase (catalyzing the reductive formation of glutamate from glutamine and 2-oxo-glutarate) restored the growth characteristics while reducing glycerol accumulation and increasing ethanol formation.261

2. Deleting the gene (FSP1) encoding the transporter protein for glycerol also improved ethanol production while decreasing the accumulation of glycerol, acetate, and pyruvate—presumably increasing the intracellular concentration of glycerol (by blocking its export) accelerates its metabolism by a reversal of the glycerol kinase and glycerol 3-phosphate dehydrogenase reactions.262

3. Improved production of ethanol was also achieved by deleting FPS1 and overexpressing GLTl, accompanied by reduced accumulation of glycerol, acetate, and pyruvate.263

A more subtle metabolic conundrum may limit ethanol accumulation in Z. mobilis: although the cells are internally “unstructured” prokaryotes, lacking membrane — limited intracellular organelles, different redox microenvironments may occur in which two forms of ADH (rather than one) could simultaneously catalyze ethanol synthesis and oxidation, setting up a futile cycle; this has only been demonstrated under aerobic growth conditions, but the functions of the two ADH enzymes (one Zn-dependent, the other Fe-dependent) inside putative redox microcompartments within the cell warrant further investigation.264

S. cerevisiae, along with E. coli, B. subtilis, and a few other organisms, formed the initial “Rosetta Stone” for genome research, providing a well-characterized group of genes for comparative sequence analysis; coding sequences of other organisms lacking detailed knowledge of their biochemistry could be assigned protein func­tionalities based on their degree of homology to known genes. Genome-scale meta­bolic networks can now be reconstructed and their properties examined in computer simulations of biotechnological processes. Such models can certainly generate good fits to experimental data for the rate of growth, glucose utilization, and (under micro — aerobiosis) ethanol formation but have limited predictive value.265 One limitation is that the biosynthetic genes comprise too conservative a functional array, lacking the sensing mechanisms and signaling pathways that sense and respond to nutri­tional status and other environmental factors.266 These elements function to estab­lish developmental and morphological programs in the wild-type ecology and have unpredictable effects on growth patterns in the fluctuating nutritional topologies of large imperfectly mixed industrial fermentors. If computer-aided design for metabo­lism is to be feasible for industrially relevant microbes, added sophistications must be incorporated — equally, however, radical restructuring is necessary to accommo­date the gross differences in metabolic patterns between bacteria such as E. coli and, for example, the Entner-Doudoroff pathway organisms or when P. stipitis metabo­lism is compared in detail with the far better characterized pathway dynamics in S. cerevisiae.267,268 Comparing metabolic profiles in 14 yeast species revealed many important similarities with S. cerevisiae, but Pichia augusta may possess the ability to radically rebalance the growth demand for NADPH from the pentose phosphate pathway (figure 3.2); this suggests a transhydrogenase activity that could be used to rebalance NADH/NADPH redox cofactors for xylose metabolism (section 3.2.1).269

In the immediate and short-term future, therefore, applied genomics can suc­cessfully define the impacts of more focused changes in quantitative gene expression. Developing a kinetic model of the pentose phosphate and Entner-Doudoroff pathways in Z. mobilis, and comparing the predictions with experimental results, showed that the activities of xylose isomerase and transaldolase were of the greatest significance for ethanol production but that overexpressing the enzymic link between the two pathways (phosphoglucose isomerase) was unnecessary.270 Such systematic analysis can help not only in maximizing fermentation efficiencies but also in minimizing the degree of expression of heterologous enzymes to reduce “metabolic burdens” in recombinant strains. Although in silico predictions failed to account for contin­ued xylitol accumulation by a recombinant, xylose-utilizing S. cerevisiae strain on xylose (suggesting continued problems in balancing cofactor requirement in reduc — tase/dehydrogenase reactions and/or still unidentified limitations to later catabolic steps in xylose consumption), computer simulations did accurately highlight glycerol formation as a key target for process improvement: expressing a nonphosphorylating NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase increased carbon flow to pyruvate (rather than being diverted to glycerol formation), reduced glycerol accumulation, and improved ethanol production by up to 25%.271272