Background Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/ T7 and XylS/ Pm promoters allowed optimisation of their expression levels. Conclusion Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced ethyl acetate from glucose at an unprecedented level, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

Background Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in Wickerhamomyces anomalus. Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as E. coli, expression of heterologous proteins with eukaryotic signal sequences may not be optimal. Results Unprocessed and synthetically truncated eat1 variants of Kluyveromyces marxianus and Wickerhamomyces anomalus have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in E. coli. Conclusion By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in E. coli. Truncated variants of eat1 could therefore benefit future engineering approaches towards efficient ethyl acetate production.

Sustainable production of bulk chemicals is one of the major challenges in the chemical industry, particularly due to their low market prices. This includes short and medium chain esters, which are used in a wide range of applications, for example fragrance compounds, solvents, lubricants or biofuels. However, these esters are produced mainly through unsustainable, energy intensive processes. Microbial conversion of biomass-derived sugars into esters may provide a sustainable alternative. This review provides a broad overview of natural ester production by microorganisms. The underlying ester-forming enzymatic mechanisms are discussed and compared, with particular focus on alcohol acyltransferases (AATs). This large and versatile group of enzymes condense an alcohol and an acyl-CoA to form esters. Natural production of esters typically cannot compete with existing petrochemical processes. Much effort has therefore been invested in improving in vivo ester production through metabolic engineering. Identification of suitable AATs and efficient alcohol and acyl-CoA supply are critical to the success of such strategies and are reviewed in detail. The review also focusses on the physical properties of short and medium chain esters, which may simplify downstream processing, while limiting the effects of product toxicity. Furthermore, the esters could serve as intermediates for the synthesis of other compounds, such as alcohols, acids or diols. Finally, the perspectives and major challenges of microorganism-derived ester synthesis are presented.

Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.

Esters are essential for the flavor and aroma of fermented products, and are mainly produced by alcohol acyl transferases (AATs). A recently discovered AAT family named Eat (Ethanol acetyltransferase) contributes to ethyl acetate synthesis in yeast. However, its effect on the synthesis of other esters is unknown. In this study, the role of the Eat family in ester synthesis was compared to that of other Saccharomyces cerevisiae AATs (Atf1p, Atf2p, Eht1p, and Eeb1p) in silico and in vivo. A genomic study in a collection of industrial S. cerevisiae strains showed that variation of the primary sequence of the AATs did not correlate with ester production. Fifteen members of the EAT family from nine yeast species were overexpressed in S. cerevisiae CEN.PK2-1D and were able to increase the production of acetate and propanoate esters. The role of Eat1p was then studied in more detail in S. cerevisiae CEN.PK2-1D by deleting EAT1 in various combinations with other known S. cerevisiae AATs. Between 6 and 11 esters were produced under three cultivation conditions. Contrary to our expectations, a strain where all known AATs were disrupted could still produce, e.g., ethyl acetate and isoamyl acetate. This study has expanded our understanding of ester synthesis in yeast but also showed that some unknown ester-producing mechanisms still exist.

A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175g/l lactic acid at pH 2.8. Its genome and transcriptomes were sequenced and annotated. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190g/l at pH 3.8 and 129g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH.

Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains.

The tricarboxylic acid (TCA) cycle has been used for decades in the microbial production of chemicals such as citrate, L-glutamate, and succinate. Maximizing yield is key for cost-competitive production. However, for most TCA cycle products, the maximum pathway yield is lowerthan the theoretical maximum yield (YE). For succinate, this was solved by creating two pathways to the product, using both branches of the TCA cycle, connected by the glyoxylate shunt (GS). A similar solution cannot be applied directly for production of compounds from the oxidative branch of the TCA cycle because irreversible reactions are involved. Here, we describe how this can be overcome and what the impact is on the yield.

Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L.

Itaconic acid, an unsaturated C5-dicarboxylic acid, is a biobased building block for the polymer industry. The purpose of this study was to establish proof of principle for an anaerobic fermentation process for the production of itaconic acid by modification of the mixed acid fermentation pathway of E. coli. E. coli BW25113 (DE3) and the phosphate acetyltransferase (pta) and lactate dehydrogenase (ldhA) deficient strain E. coli BW25113 (DE3) Δpta-ΔldhA were used to study anaerobic itaconate production in E. coli. Heterologous expression of the gene encoding cis-aconitate decarboxylase (cadA) from A. terreus in E. coli BW25113 (DE3) did not result in itaconate production under anaerobic conditions, but 0.08 mM of itaconate was formed when the genes encoding citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum were also expressed. The same amount was produced when cadA was expressed in E. coli BW25113 (DE3) Δpta-ΔldhA. The titre increased 8 times to 0.66 mM (1.2 % Cmol) when E. coli BW25113 (DE3) Δpta-ΔldhA also expressed gltA and acnA. In addition, this strain produced 8.5 mM (13 % Cmol) of glutamate. The use of a nitrogen-limited growth medium reduced the accumulation of glutamate by nearly 50 % compared to the normal medium, and also resulted in a more than 3-fold increase of the itaconate titre to 2.9 mM. These results demonstrated that E. coli has potential to produce itaconate and glutamate under anaerobic conditions, closing the redox balance by co-production of succinate or ethanol with H2 and CO2.

Interest in sustainable development has led to efforts to replace petrochemical-based monomers with biomass-based ones. Itaconic acid, a C5-dicarboxylic acid, is a potential monomer for the chemical industry with many prospective applications. cis-aconitate decarboxylase (CadA) is the key enzyme of itaconate production, converting the citric acid cycle intermediate cis-aconitate into itaconate. Heterologous expression of cadA from Aspergillus terreus in Escherichia coli resulted in low CadA activities and production of trace amounts of itaconate on Luria-Bertani (LB) medium (<10 mg/L). CadA was primarily present as inclusion bodies, explaining the low activity. The activity was significantly improved by using lower cultivation temperatures and mineral medium, and this resulted in enhanced itaconate titres (240 mg/L). The itaconate titre was further increased by introducing citrate synthase and aconitase from Corynebacterium glutamicum and by deleting the genes encoding phosphate acetyltransferase and lactate dehydrogenase. These deletions in E. coli’s central metabolism resulted in the accumulation of pyruvate, which is a precursor for itaconate biosynthesis. As a result, itaconate production in aerobic bioreactor cultures was increased up to 690 mg/L. The maximum yield obtained was 0.09 mol itaconate/mol glucose. Strategies for a further improvement of itaconate production are discussed.

BACKGROUND:Genome scale metabolic reconstructions are developed to efficiently engineer biocatalysts and bioprocesses based on a rational approach. However, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. Pseudomonas putida KT2440 is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of NADH and NAD. To apply this strain optimally in industrial production, a previously developed genome-scale metabolic model (iJP815) was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches.RESULTS:To substantially improve the accuracy of the genome scale model (iJP815), continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. Besides, the maximum biomass yield (on substrate) of 0.397 gDCW · gglc-1, the maintenance coefficient of 0.037 gglc · gDCW-1 · h-1 and the maximum specific growth rate of 0.59 h-1 were determined. Only the DNA fraction increased with the specific growth rate. This resulted in reliable estimation for the Growth-Associated Maintenance (GAM) of 85 mmolATP · gDCW-1 and the Non Growth-Associated Maintenance (NGAM) of 3.96 mmolATP · gDCW-1 · h-1. Both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed. Contrasting already published 13C flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. Furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links.CONCLUSIONS:By careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of Pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. By consideration of substrate specific variation in stoichiometry and kinetics, it can be extended to other substrates and new mutants.

Of the many ways hydrogen can be produced, this chapter focuses on biological hydrogen production by thermophilic bacteria and archaea in dark fermentations. The thermophiles are held as promising candidates for a cost-effective fermentation process, because of their relatively high yields and broad substrate palette. Yet many challenges remain to be faced, including improving productivity, tolerance to high osmolality and growth inhibitors, and reactor configuration. This review consolidates current insights in the quest for high yields and productivities within thermophilic hydrogen production. Important is to understand how environmental parameters affect the redox-and energy metabolism of the microorganism(s) involved. This knowledge is required for designing an optimal bioreactor configuration and operation.

Pseudomonas putida KT2440‐JD1 is able to cometabolize benzoate to cis, cis‐muconate in the presence of glucose as growth substrate. P. putida KT2440‐JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis‐muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis‐muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis‐muconate. To prevent overdoses of benzoate, a pH‐stat fed‐batch process for the production of cis, cis‐muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH‐stat fed‐batch process was 0.6 g (4.3 mmol) g dry cell weight −1 h −1 , whereas 18.5 g L −1 cis, cis‐muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH‐stat fed‐batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motility. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup(-) mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH4+ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H-2/mol sucrose) and maximum volumetric productivity (7.1 mmol H-2/L-c.h) were obtained in the absence of NH4+. The effluent of the dark fermentation containing no NH4+ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H-2/L-c.h) were attained using the hup(-) mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H-2/mol sucrose in dark fermentation to 13.7 mol H-2/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H-2/mole of sucrose) by sequential dark and photofermentation. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

BACKGROUND:Using a functional genomics approach we addressed the impact of folate overproduction on metabolite formation and gene expression in Lactobacillus plantarum WCFS1. We focused specifically on the mechanism that reduces growth rates in folate-overproducing cells.RESULTS:Metabolite formation and gene expression were determined in a folate-overproducing- and wild-type strain. Differential metabolomics analysis of intracellular metabolite pools indicated that the pool sizes of 18 metabolites differed significantly between these strains. The gene expression profile was determined for both strains in pH-regulated chemostat culture and batch culture. Apart from the expected overexpression of the 6 genes of the folate gene cluster, no other genes were found to be differentially expressed both in continuous and batch cultures. The discrepancy between the low transcriptome and metabolome response and the 25% growth rate reduction of the folate overproducing strain was further investigated. Folate production per se could be ruled out as a contributing factor, since in the absence of folate production the growth rate of the overproducer was also reduced by 25%. The higher metabolic costs for DNA and RNA biosynthesis in the folate overproducing strain were also ruled out. However, it was demonstrated that folate-specific mRNAs and proteins constitute 8% and 4% of the total mRNA and protein pool, respectively.CONCLUSION:Folate overproduction leads to very little change in metabolite levels or overall transcript profile, while at the same time the growth rate is reduced drastically. This shows that Lactobacillus plantarum WCFS1 is unable to respond to this growth rate reduction, most likely because the growth-related transcripts and proteins are diluted by the enormous amount of gratuitous folate-related transcripts and proteins.