Global Transcription Machinery Engineering Research Articles

Isobutanol tolerance and production of Saccharomyces cerevisiae can be improved by engineering its TATA-binding protein Spt15.

Low isobutanol tolerance of Saccharomyces cerevisiae limits its application in isobutanol fermentation. Here, we used global transcription machinery engineering to screen mutants with higher isobutanol tolerance and elevated isobutanol titres. TATA-binding protein Spt15 was used as the target of global transcription machinery engineering for improvement of such complex phenotypes. A random mutagenesis library of S. cerevisiae TATA-binding protein Spt15 was constructed and subjected to screening under isobutanol stress. A mutant strain (denoted as spt15-3) with improved isobutanol tolerance was identified. There were three mutations of Spt15 in strain spt15-3, including deletion of A at position -132 nt upstream of initiation codon, insertion of G at position -65 nt upstream of initiation codon and a synonymous mutation at position 315 nt (T → C) downstream of initiation codon. We then metabolically engineered isobutanol synthesis in strains harbouring plasmids YCplac22 containing these Spt15 mutations. Delta integration was used to overexpress ILV3 gene, and 2μ plasmids carrying PGK1p-ILV2 and PGK1p-ARO10 were used to overexpress ILV2 and ARO10 genes. After 24-h micro-aerobic fermentation, Engi-3 produced 0·556 g l-1 isobutanol, which was 404% and 25·3% greater than isobutanol produced by control Engi-1 and engineered Engi-2, respectively. After 28 h, Engi-4 produced 0·459 g l-1 isobutanol, which was 315% and 3·2% greater than isobutanol produced Engi-1 and Engi-2, respectively. RNA-Seq-based transcriptome analysis shows that mutations of Spt15 in strain spt15-3 increased the expression of SPT15. Meanwhile, compared with strain Engi-3, the spt15-3 mutation downregulated the expression of genes involved in the TCA cycle and glyoxylic acid cycle, but increased the expression of genes related to cell stability. This work demonstrates that isobutanol tolerance and production of S. cerevisiae can be improved by engineering its TATA-binding protein Spt15. This study clarified the molecular mechanisms regulating isobutanol production and tolerance in S. cerevisiae.

Development and optimization of a microbial co-culture system for heterologous indigo biosynthesis

BackgroundIndigo is a color molecule with a long history of being used as a textile dye. The conventional production methods are facing increasing economy, sustainability and environmental challenges. Therefore, developing a green synthesis method converting renewable feedstocks to indigo using engineered microbes is of great research and application interest. However, the efficiency of the indigo microbial biosynthesis is still low and needs to be improved by proper metabolic engineering strategies.ResultsIn the present study, we adopted several metabolic engineering strategies to establish an efficient microbial biosynthesis system for converting renewable carbon substrates to indigo. First, a microbial co-culture was developed using two individually engineered E. coli strains to accommodate the indigo biosynthesis pathway, and the balancing of the overall pathway was achieved by manipulating the ratio of co-culture strains harboring different pathway modules. Through carbon source optimization and application of biosensor-assisted cell selection circuit, the indigo production was improved significantly. In addition, the global transcription machinery engineering (gTME) approach was utilized to establish a high-performance co-culture variant to further enhance the indigo production. Through the step-wise modification of the established system, the indigo bioproduction reached 104.3 mg/L, which was 11.4-fold higher than the parental indigo producing strain.ConclusionThis work combines modular co-culture engineering, biosensing, and gTME for addressing the challenges of the indigo biosynthesis, which has not been explored before. The findings of this study confirm the effectiveness of the developed approach and offer a new perspective for efficient indigo bioproduction. More broadly, this innovative approach has the potential for wider application in future studies of other valuable biochemicals’ biosynthesis.

Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories.

Creation of a Low-Alcohol-Production Yeast by a Mutated SPT15 Transcription Regulator Triggers Transcriptional and Metabolic Changes During Wine Fermentation.

There is significant interest in the wine industry to develop methods to reduce the ethanol content of wine. Here the global transcription machinery engineering (gTME) technology was used to engineer a yeast strain with decreased ethanol yield, based on the mutation of the SPT15 gene. We created a strain of Saccharomyces cerevisiae (YS59-409), which possessed ethanol yield reduced by 34.9%; this was accompanied by the increase in CO2, biomass, and glycerol formation. Five mutation sites were identified in the mutated SPT15 gene of YS59-409. RNA-Seq and metabolome analysis of YS59-409 were conducted compared with control strain, suggesting that ribosome biogenesis, nucleotide metabolism, glycolysis flux, Crabtree effect, NAD+/NADH homeostasis and energy metabolism might be regulated by the mutagenesis of SPT15 gene. Furthermore, two genes related to energy metabolism, RGI1 and RGI2, were found to be associated with the weakened ethanol production capacity, although the precise mechanisms involved need to be further elucidated. This study highlighted the importance of applying gTME technology when attempting to reduce ethanol production by yeast, possibly reprogramming yeast’s metabolism at the global level.

Improved ethanol tolerance and production of Saccharomyces cerevisiae by global transcription machinery engineering via directed evolution of the SPT8 gene

ABSTRACTEthanol tolerance involves complex interactions of several genes in the Saccharomyces cerevisiae genome that encodes proteins that are the subunits of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex, particularly those that interact with the TATA-binding protein. Global transcription machinery engineering has been used to reprogram the transcription levels of multiple genes to change cellular phenotypes important for technological applications by altering key proteins by error-prone polymerase chain reaction (epPCR) mutations. In this study, we produced S. cerevisiae mutants from the wild-type wine yeast Y01 strain by directed evolution of the SPT8 (suppressor of ty insertions 8) gene using epPCR combined with construction and overexpression of the constructed pUPST mutant library for improved ethanol tolerance and production. We found that the mutant strains gave 8.9% higher ethanol tolerance and 10.8% higher ethanol production than the wild-type. The desired phenotype resulted from the combined effect of two separate mutations in SPT8, which caused serine to replace phenylalanine (Asn156His) and glycine to replace serine (Gly585Ser) in the encoded SPT8 protein.

Engineered sigma factors increase full-length antibody expression in Escherichia coli

Escherichia coli (E. coli) is a promising platform for expression of full-length antibodies owing to its several advantages as a production host (fast growth, well characterized genetics, low manufacturing cost), however, low titers from shake flask (typically < 5 mg/L) has limited its use for production of research-grade material in antibody discovery programs. In this work, we used global transcriptional machinery engineering (gTME) with high throughput screening to increase the expression of full-length antibodies in E. coli. A library of E. coli mutants carrying mutations in the global sigma factor RpoD were generated and screened using the Bacterial Antibody Display (BAD) method for enhanced expression. RpoD mutants were isolated that resulted in full-length antibody titers of up to 130.7 ± 6.6 mg/L of shake flask culture with chaperone co-expression. These results could be useful for production of several antibodies quickly in shake flasks for characterization (e.g. antigen binding, biological function) during the early discovery phase.

Improved xylose tolerance and 2,3-butanediol production of Klebsiella pneumoniae by directed evolution of rpoD and the mechanisms revealed by transcriptomics

BackgroundThe biological production of 2,3-butanediol from xylose-rich raw materials from Klebsiella pneumoniae is a low-cost process. RpoD, an encoding gene of the sigma factor, is the key element in global transcription machinery engineering and has been successfully used to improve the fermentation with Escherichia coli. However, whether it can regulate the tolerance in K. pneumoniae remains unclear.ResultsIn this study, the kpC mutant strain was constructed by altering the expression quantity and genotype of the rpoD gene, and this exhibited high xylose tolerance and 2,3-butanediol production. The xylose tolerance of kpC strain was increased from 75 to 125 g/L, and the yield of 2,3-butanediol increased by 228.5% compared with the parent strain kpG, reaching 38.6 g/L at 62 h. The RNA sequencing results showed an upregulated expression level of 500 genes and downregulated expression level of 174 genes in the kpC mutant strain. The pathway analysis further showed that the differentially expressed genes were mainly related to signal transduction, membrane transport, carbohydrate metabolism, and energy metabolism. The nine most-promising genes were selected based on transcriptome sequencing, and were evaluated for their effects on xylose tolerance. The overexpression of the tktA encoding transketolase, pntA encoding NAD(P) transhydrogenase subunit alpha, and nuoF encoding NADH dehydrogenase subunit F conferred increased xylose consumption and increased 2,3-butanediol production to K. pneumoniae.ConclusionsThese results suggest that the xylose tolerance and 2,3-butanediol production of K. pneumoniae can be greatly improved by the directed evolution of rpoD. By applying transcriptomic analysis, the upregulation of tktA, pntA, and nuoF that were coded are essential for the xylose consumption and 2,3-butanediol production. This study will provide reference for further research on improving the fermentation abilities by means of other organisms.

Engineered global regulator H-NS improves the acid tolerance of E. coli

BackgroundAcid stress is often encountered during industrial fermentation as a result of the accumulation of acidic metabolites. Acid stress increases the intracellular acidity and can cause DNA damage and denaturation of essential enzymes, thus leading to a decrease of growth and fermentation yields. Although acid stress can be relieved by addition of a base to the medium, fermentations with acid-tolerant strains are generally considered much more efficient and cost-effective.ResultsIn this study, the global regulator H-NS was found to have significant influence on the acid tolerance of E. coli. The final OD600 of strains overexpressing H-NS increased by 24% compared to control, when cultured for 24 h at pH 4.5 using HCl as an acid agent. To further improve the acid tolerance, a library of H-NS was constructed by error-prone PCR and subjected to selection. Five mutants that conferred a significant growth advantage compared to the control strain were obtained. The final OD600 of strains harboring the five H-NS mutants was enhanced by 26–53%, and their survival rate was increased by 10- to 100-fold at pH 2.5. Further investigation showed that the improved acid tolerance of H-NS mutants coincides with the activation of multiple acid resistance mechanisms, in particular the glutamate- and glutamine-dependent acid resistance system (AR2). The improved acid tolerance of H-NS mutants was also demonstrated in media acidified by acetic acid and succinic acid, which are common acidic fermentation by-products or products.ConclusionsThe results obtained in this work demonstrate that the engineering of H-NS can enhance the acid tolerance of E. coli. More in general, this study shows the potential of the engineering of global regulators acting as repressors, such as H-NS, as a promising method to obtain phenotypes of interest. This approach could expand the spectrum of application of global transcription machinery engineering.

Engineering TATA-binding protein Spt15 to improve ethanol tolerance and production in Kluyveromyces marxianus

BackgroundLow ethanol tolerance of Kluyveromyces marxianus limits its application in high-temperature ethanol fermentation. As a complex phenotype, ethanol tolerance involves synergistic actions of many genes that are widely distributed throughout the genome, thereby being difficult to engineer. TATA-binding protein is the most common target of global transcription machinery engineering for improvement of complex phenotypes.ResultsA random mutagenesis library of K. marxianus TATA-binding protein Spt15 was constructed and subjected to screening under ethanol stress. Two mutant strains with improved ethanol tolerance were identified, one of which (denoted as M2) exhibited increased ethanol productivity. The mutant of Spt15 in strain M2 (denoted as Spt15-M2) has a single amino acid substitution at position 31 (Lys → Glu). RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from Spt15-M2. Spt15-M2 caused changes in transcriptional level of most of the genes in the central carbon metabolism network. Compared with control strain, 444 differentially expressed genes (DEGs) were identified in strain M2 (fold change > 2, Padj < 0.05), including 48 up-regulated and 396 down-regulated. The up-regulated DEGs are involved in amino acid transport, long-chain fatty acid biosynthesis and MAPK signaling pathway, while the down-regulated DEGs are related to ribosome biogenesis, translation and protein synthesis. Five candidate genes (GAP1, GNP1, FAR1, STE2 and TEC1), which were found to be up-regulated in M2 strain, were overexpressed for a gain-of-function assay. However, the overexpression of no single gene helped improve ethanol tolerance as SPT15-M2 did.ConclusionsThis work demonstrates that ethanol tolerance of K. marxianus can be improved by engineering its TATA-binding protein. A single amino acid substitution (K31E) of TATA-binding protein Spt15 is able to bring differential expression of hundreds of genes that acted as an interconnected network for the phenotype of ethanol tolerance. Future perspectives of this technique in K. marxianus were discussed.

A novel constructed SPT15 mutagenesis library of Saccharomyces cerevisiae by using gTME technique for enhanced ethanol production

During the last few years, the global transcription machinery engineering (gTME) technique has gained more attention as an effective approach for the construction of novel mutants. Genetic strategies (molecular biology methods) were utilized to get mutational for both genes (SPT15 and TAF23) basically existed in the Saccharomyces cerevisiae genome via screening the gTME approach in order to obtain a new mutant S. cerevisiae diploid strain. The vector pYX212 was utilized to transform these genes into the control diploid strain S. cerevisiae through the process of mating between haploids control strains S. cerevisiae (MAT-a [CICC 1374]) and (MAT-α [CICC 31144]), by using the oligonucleotide primers SPT15-EcoRI-FW/SPT15-SalI-RV and TAF23-SalI-FW/TAF23-NheI-RV, respectively. The resultant mutants were examined for a series of stability tests. This study showed how strong the effect of using strategic gTME with the importance of the modification we conducted in Error Prone PCR protocol by increasing MnCl2 concentration instead of MgCl2. More than ninety mutants we obtained in this study were distinguished by a high level production of bio-ethanol as compared to the diploid control strain.

Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms

BackgroundBesides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the transcription factor Haa1 is an important determinant of the tolerance to this acid. The engineering of Haa1 either by overexpression or mutagenesis has therefore been considered to be a promising avenue towards the construction of more robust strains with improved acetic acid tolerance.ResultsBy applying the concept of global transcription machinery engineering to the regulon-specific transcription factor Haa1, a mutant allele containing two point mutations could be selected that resulted in a significantly higher acetic acid tolerance as compared to the wild-type allele. The level of improvement obtained was comparable to the level obtained by overexpression of HAA1, which was achieved by introduction of a second copy of the native HAA1 gene. Dissection of the contribution of the two point mutations to the phenotype showed that the major improvement was caused by an amino acid exchange at position 135 (serine to phenylalanine). In order to further study the mechanisms underlying the tolerance phenotype, Haa1 translocation and transcriptional activation of Haa1 target genes was compared between Haa1 mutant, overproduction and wild-type strains. While the rapid Haa1 translocation from the cytosol to the nucleus in response to acetic acid was not affected in the Haa1S135F mutant strain, the levels of transcriptional activation of four selected Haa1-target genes by acetic acid were significantly higher in cells of the mutant strain as compared to cells of the wild-type strain. Interestingly, the time-course of transcriptional activation in response to acetic acid was comparable for the mutant and wild-type strain whereas the maximum mRNA levels obtained correlate with each strain’s tolerance level.ConclusionOur data confirms that engineering of the regulon-specific transcription factor Haa1 allows the improvement of acetic acid tolerance in S. cerevisiae. It was also shown that the beneficial S135F mutation identified in the current work did not lead to an increase of HAA1 transcript level, suggesting that an altered protein structure of the Haa1S135F mutant protein led to an increased recruitment of the transcription machinery to Haa1 target genes.

Elucidation of ethanol tolerance mechanisms in Saccharomyces cerevisiae by global metabolite profiling.

Ethanol, the major fermentation product of yeast, is a stress factor in yeast. We previously constructed an ethanol-tolerant mutant yeast iETS3 by using the global transcriptional machinery engineering. However, the ethanol-tolerance mechanism has not been systematically investigated. In this study, global metabolite profiling was carried out, mainly by gas chromatography/time-of-flight mass spectrometry (GC/TOF MS), to investigate the mechanisms of ethanol tolerance in iETS3. A total of 108 intracellular metabolites were identified by GC/TOF MS and high performance liquid chromatography, and these metabolites were mostly intermediates of the central carbon metabolism. The metabolite profiles of iETS3 and BY4741, cultured with or without ethanol, were significantly different based on principal component and hierarchical clustering analyses. Our metabolomic analyses identified the compositional changes in cell membranes and the activation of glutamate metabolism and the trehalose synthetic pathway as the possible mechanisms for the ethanol tolerance. These metabolic traits can be considered possible targets for further improvement of ethanol tolerance in the mutant. For example, the KGD1 deletion mutant, with up-regulated glutamate metabolism, showed increased tolerance to ethanol. This study has demonstrated that metabolomics can be a useful tool for strain improvement and phenotypic analysis of microorganisms under stress.

DNA microarray of global transcription factor mutant reveals membrane-related proteins involved in n-butanol tolerance in Escherichia coli.

BackgroundEscherichia coli has been explored as a platform host strain for biofuels production such as butanol. However, the severe toxicity of butanol is considered to be one major limitation for butanol production from E. coli. The goal of this study is therefore to construct butanol-tolerant E. coli strains and clarify the tolerance mechanisms.ResultsA recombinant E. coli strain harboring σ70 mutation capable of tolerating 2 % (v/v) butanol was isolated by the global transcription machinery engineering (gTME) approach. DNA microarrays were employed to assess the transcriptome profile of butanol-tolerant strain B8. Compared with the wild-type strain, 329 differentially expressed genes (197 up-regulated and 132 down-regulated) (p < 0.05; FC ≥ 2) were identified. These genes are involved in carbohydrate metabolism, energy metabolism, two-component signal transduction system, oxidative stress response, lipid and cell envelope biogenesis and efflux pump.ConclusionsSeveral membrane-related proteins were proved to be involved in butanol tolerance of E. coli. Two down-regulated genes, yibT and yghW, were identified to be capable of affecting butanol tolerance by regulating membrane fatty acid composition. Another down-regulated gene ybjC encodes a predicted inner membrane protein. In addition, a number of up-regulated genes, such as gcl and glcF, contribute to supplement metabolic intermediates for glyoxylate and TCA cycles to enhance energy supply. Our results could serve as a practical strategy for the construction of platform E. coli strains as biofuel producer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0527-9) contains supplementary material, which is available to authorized users.

Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis.

BackgroundWith the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important. One of the major challenges faced during the process of biofuel production is the low tolerance of the microbial host towards increasing biofuel concentrations.ResultsHere, we demonstrate that the ethanol tolerance of Zymomonas mobilis can be greatly enhanced through the random mutagenesis of global transcription factor RpoD protein, (σ70). Using an enrichment screening, four mutants with elevated ethanol tolerance were isolated from error-prone PCR libraries. All mutants showed significant growth improvement in the presence of ethanol stress when compared to the control strain. After an ethanol (9 %) stress exposure lasting 22 h, the rate of glucose consumption was approximately 1.77, 1.78 and 1.39 g L−1 h−1 in the best ethanol-tolerant strain ZM4-mrpoD4, its rebuilt mutant strain ZM4-imrpoD and the control strain, respectively. Our results indicated that both ZM4-mrpoD4 and ZM4-imrpoD consumed glucose at a faster rate after the initial 9 % (v/v) ethanol stress, as nearly 0.64 % of the initial glucose remained after 54 h incubation versus approximately 5.43 % for the control strain. At 9 % ethanol stress, the net ethanol productions by ZM4-mrpoD4 and ZM4-imrpoD during the 30–54 h were 13.0–14.1 g/l versus only 6.6–7.7 g/l for the control strain. The pyruvate decarboxylase activity of ZM4-mrpoD4 was 62.23 and 68.42 U/g at 24 and 48 h, respectively, which were 2.6 and 1.6 times higher than the control strain. After 24 and 48 h of 9 % ethanol stress, the alcohol dehydrogenase activities of ZM4-mrpoD4 were also augmented, showing an approximate 1.4 and 1.3 times increase, respectively, when compared to the control strain. Subsequent quantitative real-time PCR analysis under these stress conditions revealed that the relative expression of pdc in cultured (6 and 24 h) ZM4-mrpoD4 increased by 9.0- and 12.7-fold when compared to control strain.ConclusionsCollectively, these results demonstrate that the RpoD mutation can enhance ethanol tolerance in Z. mobilis. Our results also suggested that RpoD may play an important role in resisting high ethanol concentration in Z. mobilis and manipulating RpoD via global transcription machinery engineering (gTME) can provide an alternative and useful approach for strain improvement for complex phenotypes.

Significantly improved solvent tolerance of Escherichia coli by global transcription machinery engineering.

BackgroundEscherichia coli has emerged as a promising platform microorganism to produce biofuels and fine chemicals of industrial interests. Certain obstacles however remain to be overcome, among which organic-solvent tolerance is a crucial one.ResultsWe used global transcription machinery engineering (gTME) to improve the organic-solvent tolerance (OST) of E. coli JM109. A mutant library of σ70 encoded by rpoD was screened under cyclohexane pressure. E. coli JM109 strain harboring σ70 mutant C9 was identified with capability of tolerating 69 % cyclohexane. The rpoD mutant contains three amino-acid substitutes and a stop-codon mutation, resulting a truncated sequence containing regions σ1.1 and σ1.2. Total protein difference produced by E. coli JM109 strain harboring C9 was examined with 2D-PAGE, and 204 high-abundant proteins showed over twofold variation under different solvent stress.ConclusionsOur results show that several genes (gapA, sdhB, pepB and dppA) play critical roles in enhanced solvent tolerance of E. coli, mainly involving in maintaining higher intracellular energy level under solvent stress. Global transcription machinery engineering is therefore a feasible and efficient approach for engineering strain with enhanced OST-phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0368-4) contains supplementary material, which is available to authorized users.

Improving furfural tolerance of Zymomonas mobilis by rewiring a sigma factor RpoD protein.

Furfural from lignocellulosic hydrolysates is the key inhibitor for bio-ethanol fermentation. In this study, we report a strategy of improving the furfural tolerance in Zymomonas mobilis on the transcriptional level by engineering its global transcription sigma factor (σ(70), RpoD) protein. Three furfural tolerance RpoD mutants (ZM4-MF1, ZM4-MF2, and ZM4-MF3) were identified from error-prone PCR libraries. The best furfural-tolerance strain ZM4-MF2 reached to the maximal cell density (OD600) about 2.0 after approximately 30 h, while control strain ZM4-rpoD reached its highest cell density of about 1.3 under the same conditions. ZM4-MF2 also consumed glucose faster and yield higher ethanol; expression levels and key Entner-Doudoroff (ED) pathway enzymatic activities were also compared to control strain under furfural stress condition. Our results suggest that global transcription machinery engineering could potentially be used to improve stress tolerance and ethanol production in Z. mobilis.

A comparison of whole cell directed evolution approaches in breeding of industrial strain of Saccharomyces cerevisiae.

To reduce the fermentation cost in very high gravity fermentations of ethanol using Saccharomyces cerevisiae, whole cell directed evolution approaches were carried out. The methods used included cell ploidy manipulation, global transcription machinery engineering and genome shuffling. Ethanol production by the four methods was improved compared with the control. Notably, the ethanol yield of a strain constructed by genome shuffling was enhanced by up to 11 % more than the control reaching 120 g ethanol/l in 35 h using a very high gravity fermentation with 300 g glucose/l. Genome shuffling can create strains with improved fermentation characteristics in very high gravity fermentations.

Combinatorial and high-throughput screening approaches for strain engineering.

Microbes have long been used in the industry to produce valuable biochemicals. Combinatorial engineering approaches, new strain engineering tools derived from inverse metabolic engineering, have started to attract attention in recent years, including genome shuffling, error-prone DNA polymerase, global transcription machinery engineering (gTME), random knockout/overexpression libraries, ribosome engineering, multiplex automated genome engineering (MAGE), customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER), and library construction of "tunable intergenic regions" (TIGR). Since combinatorial approaches and high-throughput screening methods are fundamentally interconnected, color/fluorescence-based, growth-based, and biosensor-based high-throughput screening methods have been reviewed. We believe that with the help of metabolic engineering tools and new combinatorial approaches, plus effective high-throughput screening methods, researchers will be able to achieve better results on improving microorganism performance under stress or enhancing biochemical yield.

Improving industrial yeast strains: exploiting natural and artificial diversity.

Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as ‘global transcription machinery engineering’ (gTME), to induce genetic variation, providing a new source of yeast genetic diversity.

Improvement of oxidative stress tolerance in Saccharomyces cerevisiae through global transcription machinery engineering

Excessive oxidative stress poses significant damage to yeast cells during fermentation process, and finally affects fermentation efficiency and the quality of products. In this paper, global transcription machinery engineering was employed to elicit Saccharomyces cerevisiae phenotypes of higher tolerance against oxidative stress caused by H2O2. Two strains from two plasmid-based mutagenesis libraries (Spt15 and Taf25), which exhibited significant increases in oxidative stress tolerance, were successfully isolated. At moderate H2O2 shock (≤3.5 mM), a positive correlation was found between the outperformance in cell growth of the oxidation-tolerate strains and H2O2 concentration. Several mutations were observed in the native transcription factors, which resulted in a different transcriptional profile compared with the control. Catalase and superoxide dismutase activities of the two mutants increased under H2O2 stress conditions. Fermentation experiments revealed that the mutant strain taf25-3 has a shorter lag phase compared to the control one, indicating that taf25-3 had improved adaptation ability to H2O2-induced oxidative stress and higher fermentation efficiency. Our study demonstrated that several amino acid substitutions in general transcription factors (Spt15 and Taf25) could modify the cellular oxidation defense systems and improve the anti-oxidation ability of S. cerevisiae. It could make the industrial ethanol fermentation more efficient and cost-effective by using the strain of higher stress tolerance.