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Metabolic engineering for high productivity and increased robustness is needed to enable sustainable biomanufacturing of lactic acid from lignocellulosic biomass. Lactic acid is an important commodity chemical used for instance as a monomer for production of polylactic acid, a biodegradable polymer. Here, rational and model-based optimization was used to engineer a diploid, xylose fermenting Saccharomyces cerevisiae strain to produce L-lactic acid. The metabolic flux was steered towards lactic acid through the introduction of multiple lactate dehydrogenase encoding genes while deleting ERF2 , GPD1 , and CYB2. A production of 93 g/L of lactic acid with a yield of 0.84 g/g was achieved using xylose as the carbon source. To increase xylose utilization and reduce acetic acid synthesis, PHO13 and ALD6 were also deleted from the strain. Finally, CDC19 encoding a pyruvate kinase was overexpressed, resulting in a yield of 0.75 g lactic acid/g sugars consumed, when the substrate used was a synthetic lignocellulosic hydrolysate medium, containing hexoses, pentoses and inhibitors such as acetate and furfural. Notably, modeling also provided leads for understanding the influence of oxygen in lactic acid production. High lactic acid production from xylose, at oxygen-limitation could be explained by a reduced flux through the oxidative phosphorylation pathway. On the contrast, higher oxygen levels were beneficial for lactic acid production with the synthetic hydrolysate medium, likely as higher ATP concentrations are needed for tolerating the inhibitors therein. The work highlights the potential of S. cerevisiae for industrial production of lactic acid from lignocellulosic biomass. [Display omitted] • A xylose fermenting S. cerevisiae strain was engineered to produce L-lactic acid. • Rational and model-guided design was applied to increase lactic acid production. • 93 g/L of lactic acid at a yield of 0.84 g/g was produced from xylose. • A yield of 0.75 g/g was achieved in a synthetic lignocellulosic hydrolysate. • Modeling was used to understand the influence of oxygen in lactic acid production. [ABSTRACT FROM AUTHOR]

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility. [Display omitted] • Lignocellulosic carbohydrates are carbon-neutral substrates for producing fuels and chemicals. • Natural carbon catabolic pathways have been rewired in bacteria and yeasts. • The aim was to improve the efficiency of lignocellulosic carbohydrate utilization. • Strain developments by metabolic engineering has improved carbon conversion and energy balance. • Recent progress and perspectives in the use of lignocellulosic carbohydrates are discussed. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks. [ABSTRACT FROM AUTHOR]

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1 , a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1 , ISU1 , GRE3 , and IRA2 , increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products. [Display omitted] • Engineered yeast normally require respiration to convert xylose into ethanol. • Laboratory evolution created yeast that ferment xylose without respiration. • Evolved gene duplications enabled aerobic xylose fermentation. • Gene duplications and regulatory mutations enable rapid anaerobic xylose conversion. • A model proposes how xylose flux is increased and redirected by overflow metabolism. [ABSTRACT FROM AUTHOR]

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli , Saccharomyces cerevisiae , Corynebacterium glutamicum , and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells. Image 1 • Xylose can provide better cell growth, energetics, and robust supply of GDP-L-fucose for 2′-FL production. • Increased mRNA levels of 2′-FL biosynthetic enzymes enhanced 2′-FL productivity. • 2′-FL secretion capability of the engineered S. cerevisiae was different depends on the culture media. • We achieved the highest titer (25.5 g/L) among 2'-FL production by S. cerevisiae reported to date. [ABSTRACT FROM AUTHOR]

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB , and pentose phosphate pathway (PPP) genes tal - tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h−1 and 0.38 h−1, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals. • Engineered P. putida to catabolize xylose and arabinose. • Identified bottlenecks in xylose catabolism in P. putida and improved growth rate. • Max sugar consumption rate in corn stover hydrolysate was 3.3 g/L/h. • Simultaneous catabolism of glucose, xylose, arabinose, aromatics, and organic acids. [ABSTRACT FROM AUTHOR]

1,2,4-Butanetriol can be used to produce energetic plasticizer as well as several pharmaceutical compounds. Although Saccharomyces cerevisiae has some attractive characters such as high robustness for industrial production of useful chemicals by fermentation, 1,2,4-butanetriol production by S. cerevisiae has not been reported. 1,2,4-butanteriotl is produced by an oxidative xylose metabolic pathway completely different from the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways conventionally used for xylose assimilation in S. cerevisiae. In the present study, S. cerevisiae was engineered to produce 1,2,4-butanetriol by overexpression of xylose dehydrogenase (XylB), xylonate dehydratase (XylD) , and 2-ketoacid decarboxylase. Further improvement of the recombinant strain was performed by the screening of optimal 2-ketoacid decarboxylase suitable for 1,2,4-butanetriol production and the enhancement of Fe uptake ability to improve the XylD enzymatic activity. Eventually, 1.7 g/L of 1,2,4-butanetriol was produced from 10 g/L xylose with a molar yield of 24.5%. Furthermore, 1.1 g/L of 1,2,4-butanetriol was successfully produced by direct fermentation of rice straw hydrolysate. Image 1 • 2-Ketoacid decarboxylase is a key enzyme for 1,2,4-butanetriol bio-production by S. cerevisiae. • Combination of BOL2 deletion and truncated TYW1 overexpression greatly improved iron-sulfur protein XylD activity in yeast. • An engineered yeast strain efficiently produced 1.1 g/L of 1,2,4-butanetriol from xylose enriched lignocellulosic hydrolysate. [ABSTRACT FROM AUTHOR]

The most prevalent xylose-assimilating pathways in recombinant Saccharomyces cerevisiae , i.e. the xylose isomerase (XI) and the xylose reductase/xylitol dehydrogenase (XR/XDH) pathways, channel the carbon flux through the pentose phosphate pathway and further into glycolysis. In contrast, the oxidative and non-phosphorylative bacterial Weimberg pathway channels the xylose carbon through five steps into the metabolic node α-ketoglutarate (αKG) that can be utilized for growth or diverted into production of various metabolites. In the present study, steps preventing the establishment of a functional Weimberg pathway in S. cerevisiae were identified. Using an original design where a S. cerevisiae strain was expressing the essential four genes of the Caulobacter crescentus pathway (xylB, xylD, xylX, xylA) together with a deletion of FRA2 gene to upregulate the iron-sulfur metabolism, it was shown that the C. crescentus αKG semialdehyde dehydrogenase, XylA was not functional in S. cerevisiae. When replaced by the recently described analog from Corynebacterium glutamicum, KsaD, significantly higher in vitro activity was observed but the strain did not grow on xylose. Adaptive laboratory evolution (ALE) on a xylose/glucose medium on this strain led to a loss of XylB, the first step of the Weimberg pathway, suggesting that ALE favored minimizing the inhibiting xylonate accumulation by restricting the upper part of the pathway. Therefore three additional gene copies of the lower Weimberg pathway (XylD, XylX and KsaD) were introduced. The resulting S. cerevisiae strain (ΔΔfra2, xylB, 4x (xylD-xylX-ksaD)) was able to generate biomass from xylose and Weimberg pathway intermediates were detected. To our knowledge this is the first report of a functional complete Weimberg pathway expressed in fungi. When optimized this pathway has the potential to channel xylose towards value-added specialty chemicals such as dicarboxylic acids and diols. • Growth on xylose via the Weimberg pathway established in S. cerevisiae. • >50% of the xylose carbon channeled through the Weimberg pathway. • XylA identified as a bottleneck when using the pathway gene from C. crescentus. • C. glutamicum ksaD expression gives higher enzyme activity than C. crescentus xylA. • S. cerevisiae growth on xylose requires fine-tuning of Weimberg pathway enzymes. [ABSTRACT FROM AUTHOR]

Abstract Overcoming carbon catabolite repression presents a significant challenge, largely due to the complex regulatory networks governing substrate catabolism, even in microbial cells. In this work, we have engineered an E. coli strain, which we have named X2G, that not only exhibits a reversed substrate preference for xylose over glucose, but also demonstrates an unusual ability to produce significant amounts of glucose. We obtained this non-intuitive phenotype by deleting four genes in upper central metabolism: ptsI , glk , pfkA , and zwf , which respectively encode Enzyme I of the phosphotransferase system, glucokinase, the dominant isozyme of phosphofructokinase, and glucose-6-phosphate dehydrogenase. The deletion of ptsI and glk blocks glucose uptake in E. coli , while the deletion of pfkA and zwf prevents the reassimilation of carbons through glycolysis and the oxidative pentose phosphate pathway, respectively. Our strain X2G is capable of converting 34% of the carbon it takes up as xylose into exported glucose. This corresponds to a glucose production rate of 1.4 ± 0.3 mmol/g DW /h at a specific growth rate of 0.25 ± 0.03 h−1, or about 1.8 ± 0.1 mM of glucose accumulated for every unit increase in OD 600. Despite a 22% decrease in xylose uptake rate, a 33% decrease in biomass yield, and a 52% decrease in acetate production rate relative to the wild-type, the intracellular flux profile and cofactor allocation of X2G remain largely unperturbed, as elucidated through 13C-metabolic flux analysis. Further quantification of the pool sizes of key intracellular metabolites revealed that glucose secretion by X2G is likely driven by the substantial accumulation of intracellular glucose 6-phosphate, fructose 6-phosphate, glucose and fructose at levels greater than 20x of that in wild-type E. coli. Combined, our results shed light on the flexibility of central metabolism, and the opportunities this affords for producing value-added pentose- and hexose-derived products from lignocellulosic feedstocks. Graphical abstract fx1 Highlights • E. coli X2G strain was engineered to preferentially utilize xylose and secrete glucose. • Deletion of only four genes was required. • E. coli X2G converts more than 1/3 of xylose carbon atoms into secreted glucose. • 13C-Flux analysis reveals that cofactor allocation was not dramatically altered. [ABSTRACT FROM AUTHOR]

Abstract The upgrade of D -xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo -Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles , Thermotoga maritima , and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20 mM xylose to 16.1 mM inositol with a conversion efficiency of 96.6% at 70 °C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70 °C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production. Graphical abstract fx1 Highlights • An artificial 12-enzyme pathway was validated to convert xylose to inositol. • Eleven hypthermophilic enzymes were used to implement high-yield conversion. • The Maillard reaction was mitigated by optimization of reaction conditions. • The coproduction of inositol from wood sugar improves economics of biorefineries. [ABSTRACT FROM AUTHOR]

Abstract Monoethylene glycol (MEG) is an important commodity chemical with applications in numerous industrial processes, primarily in the manufacture of polyethylene terephthalate (PET) polyester used in packaging applications. In the drive towards a sustainable chemical industry, bio-based production of MEG from renewable biomass has attracted growing interest. Recent attempts for bio-based MEG production have investigated metabolic network modifications in Escherichia coli , specifically rewiring the xylose assimilation pathways for the synthesis of MEG. In the present study, we examined the suitability of Saccharomyces cerevisiae , a preferred organism for industrial applications, as platform for MEG biosynthesis. Based on combined genetic, biochemical and fermentation studies, we report evidence for the existence of an endogenous biosynthetic route for MEG production from D -xylose in S. cerevisiae which consists of phosphofructokinase and fructose-bisphosphate aldolase, the two key enzymes in the glycolytic pathway. Further metabolic engineering and process optimization yielded a strain capable of producing up to 4.0 g/L MEG, which is the highest titer reported in yeast to-date. Highlights • Monoethylene glycol (MEG) is a commodity chemical used in packaging applications. • A native MEG biosynthetic route is identified in Saccharomyces cerevisiae. • This native pathway consists of two key enzymes of glycolysis. • The disruption of xylulose kinase activity enhances MEG yield from this pathway. • Metabolic engineering and process optimization Lled to a process yielding 4 g/L MEG. [ABSTRACT FROM AUTHOR]

Abstract Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf , pfkA , glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ± 0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ± 0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ± 0.85 g/L GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ± 2.11 g/L and 1.17 ± 0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories. Highlights • A xylose induced CRISPRi system was constructed in B. subtilis. • The efficient synergetic utilization of glucose and xylose was achieved. • The first report of utilization of xylose and glucose mixtures for GlcNAc production. • 103.1 ± 2.11 g/L GlcNAc was produced in 3-L fed-batch bioreactor. [ABSTRACT FROM AUTHOR]

Abstract This paper describes how Rule Input Network Generator (RING), a network generation computational tool, can be adopted to generate a variety of complex biochemical reaction networks. The reaction language incorporated in RING allows representation of chemical compounds in biological systems with various structural complexity. Complex molecules such as oligosaccharides in glycosylation pathways can be described using a simplified representation of their monosaccharide building blocks and glycosidic bonds. The automated generation and topological network analysis features in RING also allow for: (1) constructing biochemical reaction networks in a rule-based manner, (2) generating graphical representations of the networks, (3) querying molecules containing a particular structural pattern, (4) finding the shortest synthetic pathways to a user-specified species, and (5) performing enzyme knockout to study their effect on the reaction network. Case studies involving three biochemical reaction systems: (1) Synthesis of 2-ketoglutarate from xylose in bacterial cells, (2) N-glycosylation in mammalian cells, and (3) O-glycosylation in mammalian cells are presented to demonstrate the capabilities of RING for robust and exhaustive network generation and the advantages of its post-processing features. Highlights • A generic framework for biochemical reaction network generation is proposed. • Implemented a simple representation for complex biochemical molecules. • Identified synthetic pathways to species-of-interest. • Demonstrated structure-based classification of glycans. • Presented a graphical representation of generated reaction networks. [ABSTRACT FROM AUTHOR]

Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D -xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in β-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D -glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing. [ABSTRACT FROM AUTHOR]

Malate is regarded as one of the key building block chemicals which can potentially be produced from biomass at a large scale. Although glucose has been extensively studied as the substrate for malate production, its high price and potential competition with food production are serious limiting factors. In this study, Escherichia coli was metabolically engineered to effectively produce malate from xylose, the second most abundant sugar component of lignocellulosic biomass. First, the biosynthetic route of malate was constructed by overexpressing D -tagatose 3-epimerase, L-fuculokinase, L-fuculose-phosphate aldolase, and aldehyde dehydrogenase A. Second, genes encoding malic enzyme, malate dehydrogenase, and fumarate hydratase were knocked out to eliminate malate consumption, resulting in a titer of 1.99 g/l malate and a yield of 0.47 g malate/g xylose. Third, glycolate oxidase and malate synthase were overexpressed to strengthen the conversion of glycolate to malate, which led to a titer of 4.33 g/l malate and a yield of 0.83 g malate/g xylose, reaching 93% of the theoretical yield. Finally, catalase HPII was overexpressed to decompose H 2 O 2 and alleviate its toxicity, which improved cell growth and further boosted malate titer to 5.90 g/l with a yield of 0.80 g malate/g xylose. To the best of our knowledge, this is the first study to report efficient malate production from xylose as the carbon source. [ABSTRACT FROM AUTHOR]

One of the most important research subjects of metabolic engineering is the pursuit of balanced metabolic pathways, which requires the modulation of expression of many genes. However, simultaneously modulating multiple genes on the chromosome remains challenging in prokaryotic organisms, including the industrial workhorse - Escherichia coli . In this work, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique was developed to simultaneously modulate the expression of multiple genes on the chromosome. To implement it, two plasmids were employed to target Cas9 to regulatory sequences of pathway genes, and a donor DNA plasmid library was constructed containing a regulator pool to modulate the expression of these genes. A modularized plasmid construction strategy was used to enable the assembly of a complex donor DNA plasmid library. After genome editing using this technique, a combinatorial library was obtained with variably expressed pathway genes. As a demonstration, the CFPO technique was applied to the xylose metabolic pathway genes in E. coli to improve xylose utilization. Three transcriptional units containing a total of four genes were modulated simultaneously with 70% efficiency, and improved strains were selected from the resulting combinatorial library by growth enrichment. The best strain, HQ304, displayed a 3-fold increase of the xylose-utilization rate. Finally, the xylose-utilization pathway of HQ304 was analyzed enzymologically to determine the optimal combination of enzyme activities. [ABSTRACT FROM AUTHOR]

3-Hydroxy-γ-butyrolactone (3HBL) is an attractive building block owing to its broad applications in pharmaceutical industry. Currently, 3HBL is commercially produced by chemical routes using petro-derived carbohydrates, which involves hazardous materials and harsh processing conditions. Only one biosynthetic pathway has been reported for synthesis of 3HBL and its hydrolyzed form 3,4-dihydroxybutyric acid (3,4-DHBA) using glucose and glycolic acid as the substrates and coenzyme A as the activator, which involves multiple steps (>10 steps) and suffers from low productivity and yield. Here we established a novel five-step biosynthetic pathway for 3,4-DHBA generation from D -xylose based on the non-phosphorylative D -xylose metabolism, which led to efficient production of 3,4-DHBA in Escherichia coli . Pathway optimization by incorporation of efficient enzymes for each step and host strain engineering by knocking out competing pathways enabled 1.27 g/L 3,4-DHBA produced in shake flasks, which is the highest titer reported so far. The novel pathway established in engineered E. coli strain demonstrates a new route for 3,4-DHBA biosynthesis from xylose, and this engineered pathway has great potential for industrial biomanufacturing of 3,4-DHBA and 3HBL. [ABSTRACT FROM AUTHOR]

Establishing novel synthetic routes for microbial production of chemicals often requires overcoming pathway bottlenecks by tailoring enzymes to enhance bio-catalysis or even achieve non-native catalysis. Diol dehydratases have been extensively studied for their interactions with C2 and C3 diols. However, attempts on utilizing these insights to enable catalysis on non-native substrates with more than two hydroxyl groups have been plagued with low efficiencies. Here, we rationally engineered the Klebsiella oxytoca diol dehydratase to enable and enhance catalytic activity toward a non-native C4 triol, 1,2,4-butanetriol. We analyzed dehydratase's interaction with 1,2-propanediol and glycerol, which led us to develop rationally conceived hypotheses. An in silico approach was then developed to identify and screen candidate mutants with desired activity. This led to an engineered diol dehydratase with nearly 5 fold higher catalytic activity toward 1,2,4-butanetriol than the wild type as determined by in vitro assays. Based on this result, we then expanded the 1,2,4-butanetriol pathway to establish a novel 1,4-butanediol production platform. We engineered Escherichia coli 's xylose catabolism to enhance the biosynthesis of 1,2,4-butanetriol from 224 mg/L to 1506 mg/L. By introducing the complete pathway in the engineered strain we achieve de novo biosynthesis of 1,4-butanediol at 209 mg/L from xylose. This work expands the repertoire of substrates catalyzed by diol dehydratases and serves as an elucidation to establish novel biosynthetic pathways involving dehydratase based biocatalysis. [ABSTRACT FROM AUTHOR]

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes ( xylT , xylA and xlyB ) cloned from C. acetobutylicum . Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli , until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13 C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13 C metabolic flux analysis ( 13 C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13 C-MFA using the optimal tracers [1,2- 13 C]glucose, [1,6- 13 C]glucose, [1,2- 13 C]xylose and [5- 13 C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U- 13 C]glucose and [U- 13 C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO 2 originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH 2 , NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology. [ABSTRACT FROM AUTHOR]

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study, Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae ) and 3-HP production ( pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis ( gapA A1G Δ ldhA Δ pta-ackA Δ poxB Δ glpK ). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases ( iolT1 ) and glucokinase ( glk ) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB , allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials. [ABSTRACT FROM AUTHOR]

Dicarboxylic acids are attractive biosynthetic targets due to their broad applications and their challenging manufacturing process from fossil fuel feedstock. Mesaconate is a branched, unsaturated dicarboxylic acid that can be used as a co-monomer to produce hydrogels and fire-retardant materials. In this study, we engineered nonphosphorylative metabolism to produce mesaconate from d -xylose and l -arabinose. This nonphosphorylative metabolism is orthogonal to the intrinsic pentose metabolism in Escherichia coli and has fewer enzymatic steps and a higher theoretical yield to TCA cycle intermediates than the pentose phosphate pathway. Here mesaconate production was enabled from the d -xylose pathway and the l -arabinose pathway. To enhance the transportation of d -xylose and l -arabinose, pentose transporters were examined. We identified the pentose/proton symporter, AraE, as the most effective transporter for both d -xylose and l -arabinose in mesaconate production process. Further production optimization was achieved by operon screening and metabolic engineering. These efforts led to the engineered strains that produced 12.5 g/l and 13.2 g/l mesaconate after 48 h from 20 g/l of d -xylose and l -arabinose, respectively. Finally, the engineered strain overexpressing both l -arabinose and d -xylose operons produced 14.7 g/l mesaconate from a 1:1 d -xylose and l -arabinose mixture with a yield of 85% of the theoretical maximum. (0.87 g/g). This work demonstrates an effective system that converts pentoses into a value-added chemical, mesaconate, with promising titer, rate, and yield. [ABSTRACT FROM AUTHOR]

Economically feasible bioconversion of lignocelluloses into fuels and chemicals is dependent on efficient utilization of all available sugars in lignocellulosic biomass, including hextose and pentose. Previously, we constructed a xylose fermenting strain of Saccharomyces cerevisiae through metabolic engineering and enhanced its xylose utilization capability through evolutionary engineering. However, the key mechanism of improved xylose utilization and xylose isomerase activity was not identified. In this study, we applied the concept of inverse metabolic engineering to identify the factors involved in improving xylose utilization. Genomic sequencing of the evolved strain with fast xylose utilization capability was performed, and mutations possibly affecting xylose utilization were screened. Further genetic analysis of these mutant genes revealed that mutations in ASK10 (both the site-directed mutation ASK10 M475R as well as ASK10 deletion), a stress response regulator-encoding gene, improved growth on xylose and enhanced xylose isomerase activity. We found that mutation of Ask10p did not increase xylose isomerase activity through interacting with the xylose isomerase protein or through directly regulating the xylA gene transcription. Although ASK10 deletion increased the copy number of the plasmid and improved the transcription of xylA , the site-direct mutation ASK10 M475R did not change the plasmid copy number. Interestingly, we found that both the site-directed mutation ASK10 M475R and ASK10 deletion up-regulated the transcription of molecular chaperone-encoding genes HSP26 , SSA1 and HSP104 , thereby facilitating the protein folding of xylose isomerase and enhancing xylose isomerase activity. This study revealed the important mechanism of chaperones in xylose isomerase activity regulation, and it provides valuable insights for efficient xylose metabolic strain development. [ABSTRACT FROM AUTHOR]

We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and 13 C-metabolic flux analysis ( 13 C-MFA) with [1,6- 13 C]glucose, [5- 13 C]xylose, and [1,6- 13 C]glucose+[5- 13 C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90 °C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81 °C, the maximum growth rate on glucose and xylose was 0.44 and 0.46 h −1 , respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. 13 C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, 13 C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, 13 C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain. [ABSTRACT FROM AUTHOR]

Cyanobacteria are under investigation as a means to utilize light energy to directly recycle CO 2 into chemical compounds currently derived from petroleum. Any large-scale photosynthetic production scheme must rely on natural sunlight for energy, thereby limiting production time to only lighted hours during the day. Here, an obligate photoautotrophic cyanobacterium was engineered for enhanced production of 2,3-butanediol (23BD) in continuous light, 12 h:12 h light-dark diurnal, and continuous dark conditions via supplementation with glucose or xylose. This study achieved 23BD production under diurnal conditions comparable to production under continuous light conditions. The maximum 23BD titer was 3.0 g L −1 in 10 d. Also achieving chemical production under dark conditions, this work enhances the feasibility of using cyanobacteria as industrial chemical-producing microbes. [ABSTRACT FROM AUTHOR]

The development of lignocellulose as a sustainable resource for the production of fuels and chemicals will rely on technology capable of converting the raw materials into useful compounds; some such transformations can be achieved by biological processes employing engineered microorganisms. Towards the goal of valorizing the hemicellulose fraction of lignocellulose, we designed and validated a set of pathways that enable efficient utilization of pentoses for the biosynthesis of notable two-carbon products. These pathways were incorporated into Escherichia coli , and engineered strains produced ethylene glycol from various pentoses, including simultaneously from D -xylose and L -arabinose; one strain achieved the greatest reported titer of ethylene glycol, 40 g/L, from D -xylose at a yield of 0.35 g/g. The strategy was then extended to another compound, glycolate. Using D -xylose as the substrate, an engineered strain produced 40 g/L glycolate at a yield of 0.63 g/g, which is the greatest reported yield to date. [ABSTRACT FROM AUTHOR]

The deletion of PHO13 ( pho13Δ ) in Saccharomyces cerevisiae , encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ –induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae . [ABSTRACT FROM AUTHOR]

Thermophiles are increasingly used as versatile hosts in the biotechnology industry. One of the key advantages of thermophiles is the potential to achieve high rates of feedstock conversion at elevated temperatures. The recently isolated Geobacillus strain LC300 grows extremely fast on xylose, with a doubling time of less than 30 min. In the accompanying paper, the genome of Geobacillus LC300 was sequenced and annotated. In this work, we have experimentally validated the metabolic network model using parallel 13 C-labeling experiments and applied 13 C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1- 13 C], [2- 13 C], [3- 13 C], [4- 13 C], and [5- 13 C]xylose, was used for the first time. Isotopic labeling of biomass amino acids was measured by gas chromatography mass spectrometry (GC–MS). Isotopic labeling of carbon dioxide in the off-gas was also measured by an on-line mass spectrometer. The 13 C-labeling data was then rigorously integrated for flux elucidation using the COMPLETE-MFA approach. The results provided important new insights into the metabolism of Geobacillus LC300, its efficient xylose utilization pathways, and the balance between carbon, redox and energy fluxes. The pentose phosphate pathway, glycolysis and TCA cycle were found to be highly active in Geobacillus LC300. The oxidative pentose phosphate pathway was also active and contributed significantly to NADPH production. No transhydrogenase activity was detected. Results from this work provide a solid foundation for future studies of this strain and its metabolic engineering and biotechnological applications. [ABSTRACT FROM AUTHOR]

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h −1 on glucose and 1.52 h −1 on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio׳s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications. [ABSTRACT FROM AUTHOR]

Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO 2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO 2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO 2 fixation. [ABSTRACT FROM AUTHOR]

Efficient and specific transporters may enhance pentose uptake and metabolism by Saccharomyces cerevisiae . Eight heterologous sugar transporters were characterized in S. cerevisiae . The transporter Mgt05196p from Meyerozyma guilliermondii showed the highest xylose transport activity among them. Several key amino acid residues of Mgt05196p were suggested by structural and sequence analysis and characterized by site-directed mutagenesis. A conserved aromatic residue-rich motif (YFFYY, position 332–336) in the seventh trans-membrane span plays an important role in d -xylose transport activity. The phenyl ring of the residue at position 336 may take the function to prevent d -xylose from escaping during uptake. F432A and N360S mutations enhanced the d -xylose transport activities of Mgt05196p. Furthermore, mutant N360F specifically transported d -xylose without any glucose-inhibition, high lighting its potential application in constructing glucose–xylose co-fermentation strains for biomass refining. [ABSTRACT FROM AUTHOR]

After glucose, xylose is the most abundant sugar in lignocellulosic carbon sources. However, wild-type Escherichia coli is unable to simultaneously utilize both sugars due to carbon catabolite repression (CCR). In this paper, we describe GX50, an engineered strain capable of utilizing glucose and xylose simultaneously. This strain was obtained by evolving a mutant from which araC has been deleted, and in which genes required for pentose metabolism are constitutively expressed. The strain acquired four additional mutations during adaptive evolution, including intergenic mutations in the 5′-flanking region of xylA and pyrE , and missense mutations in araE (S91I) and ybjG (D99G). In contrast to wild type E. coli , GX50 rapidly converts xylose to xylitol even if glucose is available. Notably, the strain grows well when cultured on glucose, unlike some well-known CCR-insensitive mutants defective in the glucose phosphotransferase system. Our work will advance efforts to design a metabolically efficient platform strain for potential use in producing chemicals from lignocellulose. [ABSTRACT FROM AUTHOR]

1,4-Butanediol (BD) is an important chemical that is widely used in industry with an annual demand of one million metric tons. Here we report a modular development of engineered bacteria for successful BD bio-production. Using a systems engineering concept, we partitioned our development into two parts: namely BD biosynthesis and production control. The former was implemented through a de novo pathway that functions as an enzymatic reactor, while the latter was accomplished via synthetic circuits serving as genetic controllers. To facilitate development, the carbon utilizations were also partitioned into two routes. d -xylose was exclusively designated for BD production with other carbon sources utilized for cellular growth. Additionally, a quorum-sensing mechanism was exploited for production control, and the resulting strain was capable of autonomous production of BD. This study represents an example of the synergy between synthetic biology and metabolic engineering, affirming the need for deeper integration of the two fields. [ABSTRACT FROM AUTHOR]

Fermentation of xylose, a major constituent of lignocellulose, will be important for expanding sustainable biofuel production. We sought to better understand the effects of intrinsic (genotypic) and extrinsic (growth conditions) variables on optimal gene expression of the Scheffersomyces stipitis xylose utilization pathway in Saccharomyces cerevisiae by using a set of five promoters to simultaneously regulate each gene. Three-gene (xylose reductase, xylitol dehydrogenase (XDH), and xylulokinase) and eight-gene (expanded with non-oxidative pentose phosphate pathway enzymes and pyruvate kinase) promoter libraries were enriched under aerobic and anaerobic conditions or with a mutant XDH with altered cofactor usage. Through characterization of enriched strains, we observed (1) differences in promoter enrichment for the three-gene library depending on whether the pentose phosphate pathway genes were included during the aerobic enrichment; (2) the importance of selection conditions, where some aerobically-enriched strains underperform in anaerobic conditions compared to anaerobically-enriched strains; (3) improved growth rather than improved fermentation product yields for optimized strains carrying the mutant XDH compared to the wild-type XDH. [ABSTRACT FROM AUTHOR]

In this study, we describe a novel method for producing valuable chemicals from glucose and xylose in Escherichia coli . The notable features in our method are avoidance of plasmids and expensive inducers for foreign gene expression to reduce production costs; foreign genes are knocked into the chromosome, and their expression is induced with xylose that is present in most biomass feedstock. As loci for the gene knock-in, lacZYA and some pseudogenes are chosen to minimize unexpected effects of the knock-in on cell physiology. The promoter of xylF is inducible with xylose and is combined with the T7 RNA polymerase–T7 promoter system to ensure strong gene expression. This expression system was named BICES (biomass-inducible chromosome-based expression system). As examples of BICES application, 2,3-butanediol and acetoin were successfully produced from glucose and xylose, and the maximal concentrations reached 54 g L −1 [99.6% in ( R , S )-form] and 31 g L −1 , respectively. 2,3-Butanediol and acetoin are industrially important chemicals that are, at present, produced primarily through petrochemical processes. To demonstrate usability of BICES in practical situations, we produced these chemicals from a saccharified cedar solution. From these results, we can conclude that BICES is suitable for practical production of valuable chemicals from biomass. [ABSTRACT FROM AUTHOR]

Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5′-phosphates. Pyridoxal 5′-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5′-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti , were assembled to two expression cassettes and introduced into the B. subtilis chromosome. in vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14 mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54 mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis . [ABSTRACT FROM AUTHOR]