Insights into the effects of anthropogenic activities on oil reservoir microbiome and metabolic potential.

Microbial communities have long been observed in oil reservoirs, where the subsurface conditions are major drivers shaping their structure and functions. Furthermore, anthropogenic activities such as water flooding during oil production can affect microbial activities and community compositions in oil reservoirs through the injection of recycled produced water, often associated with biocides. However, it is still unclear to what extent the introduced chemicals and microbes influence the metabolic potential of the subsurface microbiome. Here we investigated an onshore oilfield in Germany (Field A) that undergoes secondary oil production along with biocide treatment to prevent souring and microbially induced corrosion (MIC). With the integrated approach of 16 S rRNA gene amplicon and shotgun metagenomic sequencing of water-oil samples from 4 production wells and 1 injection well, we found differences in microbial community structure and metabolic functions. In the injection water samples, amplicon sequence variants (ASVs) belonging to families such as Halanaerobiaceae, Ectothiorhodospiraceae, Hydrogenophilaceae, Halobacteroidaceae, Desulfohalobiaceae, and Methanosarcinaceae were dominant, while in the production water samples, ASVs of families such as Thermotogaceae, Nitrospiraceae, Petrotogaceae, Syntrophaceae, Methanobacteriaceae, and Thermoprotei were also dominant. The metagenomic analysis of the injection water sample revealed the presence of C1-metabolism, namely, genes involved in formaldehyde oxidation. Our analysis revealed that the microbial community structure of the production water samples diverged slightly from that of injection water samples. Additionally, a metabolic potential for oxidizing the applied biocide clearly occurred in the injection water samples indicating an adaptation and buildup of degradation capacity or resistance against the added biocide.

Microbial Synthesis of Human-Hormone Melatonin at Gram Scales.

Melatonin is a commercially attractive tryptophan-derived hormone. Here we describe a bioprocess for the production of melatonin using Escherichia coli to high titers. The first engineered strain produced 0.13 g/L of melatonin from tryptophan under fed-batch fermentation conditions. A 4-fold improvement on melatonin titer was further achieved by (1) protein engineering of rate-limiting tryptophan hydroxylase to improve 5-hydroxytryptophan biosynthesis and (2) chromosomal integration of aromatic-amino-acid decarboxylase to limit byproduct formation and to minimize gene toxicity to the host cell. Fermentation optimization improved melatonin titer by an additional 2-fold. Deletion of yddG, a tryptophan exporter, exhibited an additive beneficial effect. The final engineered strain produced ∼2.0 g/L of melatonin with tryptophan supplemented externally and ∼1.0 g/L with glucose as the sole carbon source for tryptophan supply. This study lays the foundation for further developing a commercial melatonin-producing E. coli strain.

Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120's Metabolism.

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts.IMPORTANCE While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

Engineered Reversal of Function in Glycolytic Yeast Promoters.

Promoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.

Exploring small-scale chemostats to scale up microbial processes: 3-hydroxypropionic acid production in S. cerevisiae.

BACKGROUND: The physiological characterization of microorganisms provides valuable information for bioprocess development. Chemostat cultivations are a powerful tool for this purpose, as they allow defined changes to one single parameter at a time, which is most commonly the growth rate. The subsequent establishment of a steady state then permits constant variables enabling the acquisition of reproducible data sets for comparing microbial performance under different conditions. We performed physiological characterizations of a 3-hydroxypropionic acid (3-HP) producing Saccharomyces cerevisiae strain in a miniaturized and parallelized chemostat cultivation system. The physiological conditions under investigation were various growth rates controlled by different nutrient limitations (C, N, P). Based on the cultivation parameters obtained subsequent fed-batch cultivations were designed. RESULTS: We report technical advancements of a small-scale chemostat cultivation system and its applicability for reliable strain screening under different physiological conditions, i.e. varying dilution rates and different substrate limitations (C, N, P). Exploring the performance of an engineered 3-HP producing S. cerevisiae strain under carbon-limiting conditions revealed the highest 3-HP yields per substrate and biomass of 16.6 %C-mol and 0.43 g gCDW-1, respectively, at the lowest set dilution rate of 0.04 h-1. 3-HP production was further optimized by applying N- and P-limiting conditions, which resulted in a further increase in 3-HP yields revealing values of 21.1 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions. The corresponding parameters favoring an increased 3-HP production, i.e. dilution rate as well as C- and P-limiting conditions, were transferred from the small-scale chemostat cultivation system to 1-L bench-top fermenters operating in fed-batch conditions, revealing 3-HP yields of 15.9 %C-mol and 0.45 g gCDW-1 under C-limiting, as well as 25.6 %C-mol and 0.50 g gCDW-1 under phosphate-limiting conditions. CONCLUSIONS: Small-scale chemostat cultures are well suited for the physiological characterization of microorganisms, particularly for investigating the effect of changing cultivation parameters on microbial performance. In our study, optimal conditions for 3-HP production comprised (i) a low dilution rate of 0.04 h-1 under carbon-limiting conditions and (ii) the use of phosphate-limiting conditions. Similar 3-HP yields were achieved in chemostat and fed-batch cultures under both C- and P-limiting conditions proving the growth rate as robust parameter for process transfer and thus the small-scale chemostat system as powerful tool for process optimization.

Coupling S-adenosylmethionine-dependent methylation to growth: Design and uses.

We present a selection design that couples S-adenosylmethionine-dependent methylation to growth. We demonstrate its use in improving the enzyme activities of not only N-type and O-type methyltransferases by 2-fold but also an acetyltransferase of another enzyme category when linked to a methylation pathway in Escherichia coli using adaptive laboratory evolution. We also demonstrate its application for drug discovery using a catechol O-methyltransferase and its inhibitors entacapone and tolcapone. Implementation of this design in Saccharomyces cerevisiae is also demonstrated.

Quantifying the Metabolome of Pseudomonas taiwanensis VLB120: Evaluation of Hot and Cold Combined Quenching/Extraction Approaches.

Absolute quantification of free intracellular metabolites is a valuable tool in both pathway discovery and metabolic engineering. In this study, we conducted a comprehensive examination of different hot and cold combined quenching/extraction approaches to extract and quantify intracellular metabolites of Pseudomonas taiwanensis (P. taiwanensis) VLB120 to provide a useful reference data set of absolute intracellular metabolite concentrations. The suitability of commonly used metabolomics tools including a pressure driven fast filtration system followed by combined quenching/extraction techniques (such as cold methanol/acetonitrile/water, hot water, and boiling ethanol/water, as well as cold ethanol/water) were tested and evaluated for P. taiwanensis VLB120 metabolome analysis. In total 94 out of 107 detected intracellular metabolites were quantified using an isotope-ratio-based approach. The quantified metabolites include amino acids, nucleotides, central carbon metabolism intermediates, redox cofactors, and others. The acquired data demonstrate that the pressure driven fast filtration approach followed by boiling ethanol quenching/extraction is the most adequate technique for P. taiwanensis VLB120 metabolome analysis based on quenching efficiency, extraction yields of metabolites, and experimental reproducibility.

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution.

L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Additional mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium. Production using the tolerant strains resulted in 37g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology.

Engineering yeast for high-level production of stilbenoid antioxidants.

Stilbenoids, including resveratrol and its methylated derivatives, are natural potent antioxidants, produced by some plants in trace amounts as defense compounds. Extraction of stilbenoids from natural sources is costly due to their low abundance and often limited availability of the plant. Here we engineered the yeast Saccharomyces cerevisiae for production of stilbenoids on a simple mineral medium typically used for industrial production. We applied a pull-push-block strain engineering strategy that included overexpression of the resveratrol biosynthesis pathway, optimization of the electron transfer to the cytochrome P450 monooxygenase, increase of the precursors supply, and decrease of the pathway intermediates degradation. Fed-batch fermentation of the final strain resulted in a final titer of 800 mg l-1 resveratrol, which is by far the highest titer reported to date for production of resveratrol from glucose. We further integrated heterologous methyltransferases into the resveratrol platform strain and hereby demonstrated for the first time de novo biosynthesis of pinostilbene and pterostilbene, which have better stability and uptake in the human body, from glucose.

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion.

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the ß-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L(-1) of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

BACKGROUND: In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. RESULTS: Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. CONCLUSIONS: In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.

Glucose-based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae.

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N-acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L-tryptophan hydroxylase, a 5-hydroxy-L-tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O-methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co-factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L(-1) in a 76h fermentation using simulated fed-batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin.

Engineering of high yield production of L-serine in Escherichia coli.

L-serine is a widely used amino acid that has been proposed as a potential building block biochemical. The high theoretical yield from glucose makes a fermentation based production attractive. In order to achieve this goal, serine degradation to pyruvate and glycine in E. coli MG1655 was prevented by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very high serine production yield (0.45 g/g glucose) during small-scale batch fermentation in minimal medium. Serine, however, was found to be highly toxic even at low concentrations to this strain, which lead to slow growth and production during fed batch fermentation, resulting in a serine production of 8.3 g/L. The production strain was therefore evolved by random mutagenesis to achieve increased tolerance towards serine. Additionally, overexpression of eamA, a cysteine/homoserine transporter was demonstrated to increase serine tolerance from 1.6 g/L to 25 g/L. During fed batch fermentation, the resulting strain lead to the serine production titer of 11.7 g/L with yield of 0.43 g/g glucose, which is the highest yield reported so far for any organism.

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via ß-alanine.

Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the ß-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of ß-alanine and its subsequent conversion into 3HP using a novel ß-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe--a quantitative approach using 13C-based metabolic flux analysis.

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying (13)C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

Bioresponsive polymers for the detection of bacterial contaminations in platelet concentrates.

Bacterial contamination of platelet concentrates (PCs) can lead to fatal transfusion transmitted diseases and is the most abundant infectious risk in transfusion medicine. The storage conditions of PCs provide a good environment for bacterial growth. The detection of these contaminations at an early stage is therefore important to avoid the transfusion of contaminated samples. In this study, bioresponsive polymer (BRP) systems were used for the detection of microorganisms in PCs. The backbone of the polymer consisted of labelled protein (casein), which was demonstrated to be degraded by pure proteases as models and by extracellular enzymes released by contaminating microorganisms. The concomitant colour change was easily visible to the naked eye. To enhance stability, the protein was cross-linked with glycidyl methacrylate (GMA). The cross-linked polymer was easier to handle but was less sensitive than the non-cross-linked material. A contamination of a PC with 10CFU/mL S. aureus was detectable after 24 hours. The visible colour reaction was quantified as a ΔE value according to the CIELab concept. A ΔE value of 21.8 was already reached after 24 hours. Hence, this simple but effective system could prevent transfusion of a contaminated PC.

Novel protease-based diagnostic devices for detection of wound infection.

A gelatinase-based device for fast detection of wound infection was developed. Collective gelatinolytic activity in infected wounds was 23 times higher (p ≤ 0.001) than in noninfected wounds and blisters according to the clinical and microbiological description of the wounds. Enzyme activities of critical wounds showed 12-fold elevated enzyme activities compared with noninfected wounds and blisters. Upon incubation of gelatin-based devices with infected wound fluids, an incubation time of 30 minutes led to a clearly visible dye release. A 32-fold color increase was measured after 60 minutes. Both matrix metalloproteinases and elastases contributed to collective gelatinolytic enzyme activity as shown by zymography and inhibition experiments. The metalloproteinase inhibitor 1,10-phenanthroline (targeting matrix metalloproteinases) and the serine protease inhibitor phenylmethlysulfonyl fluoride (targeting human neutrophil elastase) inhibited gelatinolytic activity in infected wound fluid samples by 11-37% and 60-95%, respectively. Staphylococcus aureus and Pseudomonas aeruginosa, both known for gelatinase production, were isolated in infected wound samples.

Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate.

Growth on glycerol has already been a topic of research for several yeast species, and recent publications deal with the regulatory mechanisms of glycerol assimilation by the fission yeast Schizosaccharomyces pombe. We investigated glycerol metabolism of S. pombe from a physiological point of view, characterizing growth and metabolism on a mixture of glycerol and acetate and comparing it to growth on glucose under respirative growth conditions in chemostat experiments. On glycerol/acetate mixtures, the cells grew with a maximum specific growth rate of 0.11 h(-1) where 46 % of the carbon was channeled into biomass and the key fermentation product ethanol was not detectable. (13)C-assisted metabolic flux analysis resolved substrate distributions through central carbon metabolism, proving that glycerol is used as a precursor for glycolysis, gluconeogenesis, and the pentose phosphate pathway, while acetate enters the tricarboxylic acid cycle via acetyl-CoA. Considering compartmentalization between cytosol and mitochondria in the metabolic model, we found compartmentalization of biosynthesis for the amino acids aspartate and leucine. Balancing of redox cofactors revealed an abundant production of cytosolic NADPH that must be finally regenerated via the respiratory chain shown by the simulated and measured CO2 production and oxygen consumption rates which were in good agreement.

Production of L-lysine on different silage juices using genetically engineered Corynebacterium glutamicum.

Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.