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In E. coli , the genes of the D-xylonate catabolic pathway are present in two distinct operons, yagEFG  and yjhIHG , which are part of two cryptic phages found in the genome of this bacterium. These operons are under the control of two transcription factors: XynR which acts as a repressor of yagEFG and yjhI which is an activator of yjhIHG . Although D-xylonate is known to induce these two operons, the true inducer has not been identified yet. Through the construction of biosensors based on xynR and yjhI , using syfp2 as reporter gene in combination with mutants of genes involved in D-xylonate catabolism, it has been demonstrated that the effector of these operons is 2-keto-3-deoxy-D-xylonate, which is formed by the dehydration of D-xylonate catalyzed by the dehydratases encoded by yagF and yjhG genes. Building on the finding that these two operons were also upregulated in E. coli challenged with high concentration of platform molecule 2,4-dihydroxybutyric acid, it is reported that both XynR- and YjhI-based biosensors were also responsive to the intermediate non-natural molecule 2-oxo-4-hydroxybutyric acid, with characteristic performances in terms of response threshold, sensitivity, cooperativity, and dynamic response comparable to those of the natural effector. Given that the bottlenecks in the production of 2,4-dihydroxybutyrate from C2 and C5/C6 carbon are D-threonate dehydratase and L-homoserine transaminase, respectively, which catalyze the formation of 2-oxo-4-hydroxybutyric acid from either D-threonate or homoserine, we showed that XynR and YjhI-based biosensors could be efficient tools for the screening and selection of more active enzymes producing this compound, thereby improving the production of 2,4-dihydroxybutyrate.

Lignocellulose is the most abundant biomass on earth with an annual production of about 2 × 1011 tons. It is an inedible renewable carbonaceous resource that is very rich in pentose and hexose sugars. The ability of microorganisms to use lignocellulosic sugars can be exploited for the production of biofuels and chemicals, and their concurrent biotechnological processes could advantageously replace petrochemicals’ processes in a medium to long term, sustaining the emerging of a new economy based on bio-based products from renewable carbon sources. One of the major issues to reach this objective is to rewire the microbial metabolism to optimally configure conversion of these lignocellulosic-derived sugars into bio-based products in a sustainable and competitive manner. Systems’ metabolic engineering encompassing synthetic biology and evolutionary engineering appears to be the most promising scientific and technological approaches to meet this challenge. In this review, we examine the most recent advances and strategies to redesign natural and to implement non-natural pathways in microbial metabolic framework for the assimilation and conversion of pentose and hexose sugars derived from lignocellulosic material into industrial relevant chemical compounds leading to maximal yield, titer and productivity. These include glycolic, glutaric, mesaconic and 3,4-dihydroxybutyric acid as organic acids, monoethylene glycol, 1,4-butanediol and 1,2,4-butanetriol, as alcohols. We also discuss the big challenges that still remain to enable microbial processes to become industrially attractive and economically profitable.

(L)-2,4-dihydroxybutyrate (DHB) is a versatile compound that can serve as a precursor for the synthesis of the methionine analog 2-hydroxy-4-(methylthio)butyrate and new advanced polymers. We previously implemented in Escherichia coli an artificial biosynthetic pathway for the aerobic production of DHB from glucose, which relies on the deamination of (L)-homoserine followed by the reduction of 2-oxo-4-hydroxybutyrate (OHB) and yields DHB by an enzyme-bearing NADH-dependent OHB reductase activity. Under aerobic conditions, using NADPH as a cofactor is more favorable for reduction processes. We report the construction of an NADPH-dependent OHB reductase and increased intracellular NADPH supply by metabolic engineering to improve DHB production. Key cofactor discriminating positions were identified in the previously engineered NADH-dependent OHB reductase ( E. coli malate dehydrogenase I12V:R81A:M85Q:D86S:G179D) and tested by mutational scanning. The two point mutations D34G:I35R were found to increase the specificity for NADPH by more than three orders of magnitude. Using the new OHB reductase enzyme, replacing the homoserine transaminase with the improved variant Ec.AlaC A142P:Y275D and increasing the NADPH supply by overexpressing the pntAB gene encoding the membrane-bound transhydrogenase yielded a strain that produced DHB from glucose at a yield of 0.25 mol DHB mol Glucose −1 in shake-flask experiments, which corresponds to a 50% increase compared to previous producer strains. Upon 24 h of batch cultivation of the most advanced DHB producer strain constructed in this work, a volumetric productivity of 0.83 mmol DHB L −1  h −1 was reached.

The fermentative production of the functional precursor 2,4-dihydroxybutyrate (DHB) enables sustainable synthesis of the methionine analogue hydroxy-4-(methylthio) butyrate, which is currently still produced from fossil fuels. In this work, we aimed to optimize the aerobic production of DHB from glucose through the synthetic malyl phosphate (MalP) pathway, which comprises the conversion of the natural TCA cycle intermediate malate into MalP and the subsequent reactions to yield malate semialdehyde (MalSA) and finally DHB. We first implemented the synthetic pathway in an engineered Escherichia coli strain previously reported to over-produce malate through the oxidative TCA cycle. However, DHB was only detected in trace amounts, while acetate and malate were secreted in high quantities. Subsequent construction of strains producing malate, but negligible amounts of acetate, revealed that an increased supply of malate alone is not sufficient for improved production of DHB. Instead, we discovered metabolic inefficiencies in the DHB pathway as we found that deleting the endogenous succinate semialdehyde dehydrogenase Sad, whose natural substrate is structurally similar to MalSA, strongly improved performance of the DHB pathway. Specifically, with the single knock-out of sad we could achieve a 3-fold increase in DHB production with a yield of 0.15 mol mol -1 compared to the wildtype host in shake flask experiments. With additional chromosomal expression of the mutant ppc K620S gene encoding the malate-insensitive phosphoenolpyruvate carboxylase under control of a weak constitutive promoter, we achieved a DHB yield of 0.22 mol mol -1 , which corresponds to 17% of the maximal yield under aerobic conditions.

Abstract This article reviews evolutionary engineering of Saccharomyces cerevisiae. Following a brief introduction to the ‘rational’ metabolic engineering approach and its limitations such as extensive genetic and metabolic information requirement on the organism of interest, complexity of cellular physiological responses, and difficulties of cloning in industrial strains, evolutionary engineering is discussed as an alternative, inverse metabolic engineering strategy. Major evolutionary engineering applications with S. cerevisiae are then discussed in two general categories: (1) evolutionary engineering of substrate utilization and product formation and (2) evolutionary engineering of stress resistance. Recent developments in functional genomics methods allow rapid identification of the molecular basis of the desired phenotypes obtained by evolutionary engineering. To conclude, when used alone or in combination with rational metabolic engineering and/or computational methods to study and analyze processes of adaptive evolution, evolutionary engineering is a powerful strategy for improvement in industrially important, complex properties of S. cerevisiae.

Iron plays an essential role in all organisms and is involved in the structure of many biomolecules. It also regulates the Fenton reaction where highly reactive hydroxyl radicals occur. Iron is also important for microbial biodiversity, health and nutrition. Excessive iron levels can cause oxidative damage in cells. Saccharomyces cerevisiae evolved mechanisms to regulate its iron levels. To study the iron stress resistance in S. cerevisiae, evolutionary engineering was employed. The evolved iron stress-resistant mutant “M8FE” was analysed physiologically, transcriptomically and by whole genome re-sequencing. M8FE showed cross-resistance to other transition metals: cobalt, chromium and nickel and seemed to cope with the iron stress by both avoidance and sequestration strategies. PHO84, encoding the high-affinity phosphate transporter, was the most down-regulated gene in the mutant, and may be crucial in iron-resistance. M8FE had upregulated many oxidative stress response, reserve carbohydrate metabolism and mitophagy genes, while ribosome biogenesis genes were downregulated. As a possible result of the induced oxidative stress response genes, lower intracellular oxidation levels were observed. M8FE also had high trehalose and glycerol production levels. Genome re-sequencing analyses revealed several mutations associated with diverse cellular and metabolic processes, like cell division, phosphate-mediated signalling, cell wall integrity and multidrug transporters.

Background Glycolic acid (GA) is a two-carbon hydroxyacid with applications in the cosmetic, textile, and medical industry. Microbial GA production from all sugars can be achieved by engineering the natural glyoxylate shunt. The synthetic (d)-xylulose-1 phosphate (X1P) pathway provides a complementary route to produce GA from (d)-xylose. The simultaneous operation of the X1P and glyoxylate pathways increases the theoretical GA yield from xylose by 20 %, which may strongly improve GA production from hemicellulosic hydrolysates. Results We herein describe the construction of an E. coli strain that produces GA via the glyoxylate pathway at a yield of 0.31 , 0.29 , and 0.37 g/g from glucose, xylose, or a mixture of glucose and xylose (mass ratio: 33:66 %), respectively. When the X1P pathway operates in addition to the glyoxylate pathway, the GA yields on the three substrates are, respectively, 0.39 , 0.43 , and 0.47 g/g. Upon constitutive expression of the sugar permease GalP, the GA yield of the strain which simultaneously operates the glyoxylate and X1P pathways further increases to 0.63 g/g when growing on the glucose/xylose mixture. Under these conditions, the GA yield on the xylose fraction of the sugar mixture reaches 0.75 g/g, which is the highest yield reported to date. Conclusions These results demonstrate that the synthetic X1P pathway has a very strong potential to improve GA production from xylose-rich hemicellulosic hydrolysates.

Background Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable. Results Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of ( d )-xylose isomerase, ( d )-xylulose-1-kinase, ( d )-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD . The production strain was optimized by deleting the genes encoding for ( d )-xylulose-5 kinase ( xylB ) and glycolaldehyde dehydrogenase ( aldA ), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20 g/L EG with a molar yield and productivity of 0.91 and 0.37 g/(L.h), respectively, in a controlled bioreactor under aerobic conditions. Conclusions We have demonstrated the feasibility to produce EG from ( d )-xylose via a synthetic pathway in E. coli at approximately 90 % of the theoretical yield.

Background The meiotic developmental pathway in yeast enables both differentiation of vegetative cells into haploid spores that ensure long-term survival, and recombination of the parental DNA to create genetic diversity. Despite the importance of proper metabolic regulation for the supply of building blocks and energy, little is known about the reprogramming of central metabolic pathways in meiotically differentiating cells during passage through successive developmental stages. Results Metabolic regulation during meiotic differentiation in budding yeast was analyzed by integrating information on genome-wide transcriptional activity, 26 enzymatic activities in the central metabolism, the dynamics of 67 metabolites, and a metabolic flux analysis at mid-stage meiosis. Analyses of mutants arresting sporulation at defined stages demonstrated that metabolic reprogramming is tightly controlled by the progression through the developmental pathway. The correlation between transcript levels and enzymatic activities in the central metabolism varies significantly in a developmental stage-dependent manner. The complete loss of phosphofructokinase activity at mid-stage meiosis enables a unique setup of the glycolytic pathway which facilitates carbon flux repartitioning into synthesis of spore wall precursors during the co-assimilation of glycogen and acetate. The need for correct homeostasis of purine nucleotides during the meiotic differentiation was demonstrated by the sporulation defect of the AMP deaminase mutant amd1 , which exhibited hyper-accumulation of ATP accompanied by depletion of guanosine nucleotides. Conclusions Our systems-level analysis shows that reprogramming of the central metabolism during the meiotic differentiation is controlled at different hierarchical levels to meet the metabolic and energetic needs at successive developmental stages.

Abstract Increased exposure to nickel compounds and alloys due to industrial development has resulted in nickel pollution and many pathological effects on human health. However, there is very limited information about nickel response, transport, and tolerance in eukaryotes. To investigate nickel resistance in the model eukaryote Saccharomyces cerevisiae, evolutionary engineering by batch selection under gradually increasing nickel stress levels was performed. Nickel hyper-resistant mutants that could resist up to 5.3 mM NiCl2, a lethal level for the reference strain, were selected. The mutants were also cross-resistant against iron, cobalt, zinc, and manganese stresses and accumulated more than twofold higher nickel than the reference strain. Global transcriptomic analysis revealed that 640 upregulated genes were related to iron homeostasis, stress response, and oxidative damage, implying that nickel resistance may share common mechanisms with iron and cobalt resistance, general stress response, and oxidative damage.