A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation.

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

Systematic engineering of Saccharomyces cerevisiae for D-lactic acid production with near theoretical yield.

D-lactic acid is a chiral three-carbon organic acid that can improve the thermostability of polylactic acid. Here, we systematically engineered Saccharomyces cerevisiae to produce D-lactic acid from glucose, a renewable carbon source, at near theoretical yield. Specifically, we screened D-lactate dehydrogenase (DLDH) variants from lactic acid bacteria in three different genera and identified the Leuconostoc pseudomesenteroides variant (LpDLDH) as having the highest activity in yeast. We then screened single-gene deletions to minimize the production of the side products ethanol and glycerol as well as prevent the conversion of D-lactic acid back to pyruvate. Based on the results of the DLDH screening and the single-gene deletions, we created a strain called ASc-d789M which overexpresses LpDLDH and contains deletions in glycerol pathway genes GPD1 and GPD2 and lactate dehydrogenase gene DLD1, as well as downregulation of ethanol pathway gene ADH1 using the L-methionine repressible promoter to minimize impact on growth. ASc-d789M produces D-lactic acid at a titer of 17.09 g/L in shake-flasks (yield of 0.89 g/g glucose consumed or 89% of the theoretical yield). Fed-batch fermentation resulted in D-lactic acid titer of 40.03 g/L (yield of 0.81 g/g glucose consumed). Altogether, our work represents progress towards efficient microbial production of D-lactic acid.

Unravelling Regioselectivity of Leuconostoc citreum ABK-1 Alternansucrase by Acceptor Site Engineering.

Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose-acceptor reaction-as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.

Purification and biochemical characterization of a novel glucansucrase from Leuconostoc citreum B-2.

OBJECTIVE: Although the extracellular polysaccharides have been analyzed in the previous period, the biochemical, enzymological characters and stimulation and inhibition effect on glucansucrase are not fully understood. RESULTS: After three steps purification, salting out, DEAE-Sepharose and Sephadex G-75, the final specific activity was 264.84 U/mg protein with 4.31-fold. The SDS-PAGE analysis of fraction gave a single band 170.35 kDa in the stained gel. The active band was analyzed with LC-MS/MS to identify glucansucrase. The highest coverage rate of dextransucrase from Leu. citreum (ACY92456.2) was 55.60%, the results were speculated that the glucansucrase secreted from Leu. citreum B-2 may be a novel glucansucrase. The purified enzyme was optimally active at 20-30 °C and pH 6.0-8.0. Metal ions K+, Na+, Ca2+, Mn2+, Mg2+, and Cr+ had an apparent stimulating effect on enzyme activity, especially in divalent ions Ca2+ and Mn2+, the residual activities were higher than 200%. In a reverse, Hg+, acetonitrile, SDS, salt, and guanidine expressed inhibition effect on enzyme residual activity. The KM and Vmax were detected to be 4.82 mM and 0.97 U/mg, respectively. CONCLUSION: All these data collectively indicate that B-2 glucansucrase is a novel one, which have good properties and may applied to new food areas.

Futile Encounter Engineering of the DSR-M Dextransucrase Modifies the Resulting Polymer Length.

The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains. Nuclear magnetic resonance analysis shows that this mutation influences both the dynamics of the active site and the water accessibility. Monte Carlo simulation of the elongation process allows interpretation of these results in terms of enhanced futile encounters, whereby the less effective binding increases the pool of effective seeds for the dextran chains and thereby directly determines the length distribution of the final polymers.

Performance of Leuconostoc citreum FDR241 during wheat flour sourdough type I propagation and transcriptional analysis of exopolysaccharides biosynthesis genes.

This study focused on the performance of the dextran producer Leuconostoc citreum as starter culture during 30 days of wheat flour type I sourdough propagation (back-slopping). As confirmed by RAPD-PCR analysis, the strain dominated throughout the propagation procedure, consisting of daily fermentations at 20 °C. The sourdoughs were characterized by consistent lactic acid bacteria cell density and acidification parameters, reaching pH values of 4.0 and mild titratable acidity. Carbohydrates consumption remained consistent during the propagation procedure, leading to formation of mannitol and almost equimolar amount of lactic and acetic acid. The addition of sucrose enabled the formation of dextran, inducing an increase in viscosity of the sourdough of 2-2.6 fold, as well as oligosaccharides. The transcriptional analysis based on glucosyltransferases genes (GH70) showed the existence in L. citreum FDR241 of at least five different dextransucrases. Among these, only one gene, previously identified as forming only α-(1-6) glycosidic bonds, was significantly upregulated in sourdough fermentation conditions, and the main responsible of dextran formation. A successful application of a starter culture during long sourdough back-slopping procedure will depend on the strain robustness and fermentation conditions. Transcriptional regulation of EPS-synthetizing genes might contribute to increase the efficiency of industrial processes.

Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family.

Leuconostoc citreumNRRL B-742 has been known for years to produce a highly α-(1→3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative α-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear α-(1→6) dextrans through the specific formation of α-(1→3) linkages. To date, BRS-B is the first reported α-(1→3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of α-(1→3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced byL. citreumNRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome ofLeuconostoc fallaxKCTC3537 andLactobacillus kunkeeiEFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found inL. citreumspecies. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.

Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803.

Both enantiomers of lactic acid, l-lactic acid and d-lactic acid, can be produced in a sustainable way by a photosynthetic microbial cell factory and thus from CO2, sunlight, and water. Several properties of polylactic acid (a polyester of polymerized lactic acid) depend on the controlled blend of these two enantiomers. Recently, cyanobacterium Synechocystis sp. strain PCC6803 was genetically modified to allow formation of either of these two enantiomers. This report elaborates on the d-lactic acid production achieved by the introduction of a d-specific lactate dehydrogenase from the lactic acid bacterium Leuconostoc mesenteroides into Synechocystis. A typical batch culture of this recombinant strain initially shows lactic acid production, followed by a phase of lactic acid consumption, until production "outcompetes" consumption at later growth stages. We show that Synechocystis is able to use d-lactic acid, but not l-lactic acid, as a carbon source for growth. Deletion of the organism's putative d-lactate dehydrogenase (encoded by slr1556), however, does not eliminate this ability with respect to d-lactic acid consumption. In contrast, d-lactic acid consumption does depend on the presence of glycolate dehydrogenase GlcD1 (encoded by sll0404). Accordingly, this report highlights the need to match a product of interest of a cyanobacterial cell factory with the metabolic network present in the host used for its synthesis and emphasizes the need to understand the physiology of the production host in detail.

Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae.

There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Both optical isomers, D-LA and L-LA, are required to produce stereocomplex PLA with improved properties. In this study, we developed Saccharomyces cerevisiae strains for efficient production of D-LA. D-LA production was achieved by expressing highly stereospecific D-lactate dehydrogenase gene (ldhA, LEUM_1756) from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 in S. cerevisiae lacking natural LA production activity. D-LA consumption after glucose depletion was inhibited by deleting DLD1 encoding D-lactate dehydrogenase and JEN1 encoding monocarboxylate transporter. In addition, ethanol production was reduced by deleting PDC1 and ADH1 genes encoding major pyruvate decarboxylase and alcohol dehydrogenase, respectively, and glycerol production was eliminated by deleting GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase. LA tolerance of the engineered D-LA-producing strain was enhanced by adaptive evolution and overexpression of HAA1 encoding a transcriptional activator involved in weak acid stress response, resulting in effective D-LA production up to 48.9 g/L without neutralization. In a flask fed-batch fermentation under neutralizing condition, our evolved strain produced 112.0 g/L D-LA with a yield of 0.80 g/g glucose and a productivity of 2.2 g/(L · h).

Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum.

BACKGROUND: IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme. RESULTS: Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS. CONCLUSIONS: Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.

Reversible logic gates based on enzyme-biocatalyzed reactions and realized in flow cells: a modular approach.

Reversible logic gates, such as the double Feynman gate, Toffoli gate and Peres gate, with 3-input/3-output channels are realized using reactions biocatalyzed with enzymes and performed in flow systems. The flow devices are constructed using a modular approach, where each flow cell is modified with one enzyme that biocatalyzes one chemical reaction. The multi-step processes mimicking the reversible logic gates are organized by combining the biocatalytic cells in different networks. This work emphasizes logical but not physical reversibility of the constructed systems. Their advantages and disadvantages are discussed and potential use in biosensing systems, rather than in computing devices, is suggested.

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

Production of a low calorie mandarin juice by enzymatic conversion of constituent sugars to oligosaccharides and prevention of insoluble glucan formation.

Over 99% of sucrose in mandarin juice (57.1 g/l in original juice to 428.4 g/l in concentrated juice) was enzymatically converted to glucooligosaccharides using 3 U dextransucrase/ml prepared from Leuconostoc mesenteroides at 28 °C. The oligosaccharide synthesis yields were 51 and 47% for the original and the concentrated mandarin juice, respectively. The degree of polymerization of oligosaccharides in the enzyme-modified juice was 2-7. Calories in the original and modified mandarin juice were 433 and 301 kcal/l (30.5% reduction). Compared with the original juice, the enzyme-modified juice showed 82% decrease of insoluble glucan formation by mutansucrase from Streptococcus mutans. A sensory evaluation of the juices revealed that the original and modified mandarin juices had sweetness values of 4.5 and 4.9 and the same values for overall acceptability.

Role of nutrients and environmental conditions for the production of dextransucrase from L. mesenteroides KIBGE-IB26.

The bacterial strains capable of producing dextransucrase enzyme were isolated from different fruits and vegetables sources. In primary screening, five strains were selected on the basis dextransucrase production and among them L. mesenteroides KIBGE- IB26 isolated from bottle gourd (Lagenaria Vulgaris) was selected for further studies. For the enhancement of enzyme production, different physicochemical parameters were optimized. Maximum production of dextransucrase was achieved after 06 hrs using sucrose (20.0 g/l) as a substrate at 25°C. Maximum dextransucrase production was achieved when medium pH was kept 7.5 before sterilization. In addition, medium was also supplemented with CaCl2 and K2HPO4 and maximum enzyme production was achieved at 0.0025 g/dl calcium chloride and 2.0 g/dl K2HPO4with enzyme activity of 87 DSU/ml/hr. Production of dextransucrase in shorter period of time makes this strain an attractive candidate for commercial production of dextransucrase.

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli.

In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Δpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.

Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics.

Phosphoketolases (PKs) are large thiamine pyrophosphate (TPP)-dependent enzymes playing key roles in a number of essential pathways of carbohydrate metabolism. The putative PK genes of Lactococcus lactis (Ll) and Leuconostoc mesenteroides (Lm) were cloned in a prokaryotic vector, and the encoded proteins were expressed and purified yielding high purity proteins termed PK-Ll and PK-Lm, respectively. Similarly, the PK gene of Pseudomonas aeruginosa was expressed, and the corresponding protein (PK-Pa) was purified to homogeneity. The amino acid sequences predicted on the basis of genes' nucleotide sequences were confirmed by mass spectrometry and display low relative similarities. Circular dichroism (CD) spectra of these proteins predict higher α-helix than ß-strand contents. In addition, it is predicted that PK-Ll contains tightly packed domains. Enzymatic analysis showed that all three recombinant proteins, despite their dissimilar amino acid sequences, are active PKs and accept both xylulose 5-phosphate (X5P) and fructose 6-phosphate (F6P) as substrates. However, they display substantially higher preference for X5P than for F6P. Kinetic measurements indicated that PK-Pa has the lowest Km values for X5P and F6P suggesting the highest capacity for substrate binding. PK-Ll has the largest kcat values for both substrates. Nevertheless, in terms of substrate specificity constant, PK-Pa has been found to be the most active PK against X5P. Structural models for all three analysed PKs predict similar folds in spite of amino acid sequence dissimilarities and contribute to understanding the enzymatic peculiarities of PK-Pa compared to PK-Ll and PK-Lm.

Effects of mutations at threonine-654 on the insoluble glucan synthesized by Leuconostoc mesenteroides NRRL B-1118 glucansucrase.

Twelve different amino acids were each substituted for threonine-654 in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Both the native and the cloned enzyme with threonine at position 654 produced a water-insoluble glucan containing approximately 44 mol% 1,3-disubstituted α-D-glucopyranosyl units and 29 mol% 1,6-disubstituted α-D-glucopyranosyl units. Several substitutions yielded an enzyme that produced an increased percentage of 1,3-disubstituted α-D-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted α-D-glucopyranosyl units. Only one substitution, tyrosine, resulted in a significant increase in the percentage of 1,6-disubstituted α-D-glucopyranosyl units, with a concomitant increase in glucan yield. The mutated enzymes that produced the highest levels of 1,3-disubstituted α-D-glucopyranosyl units were also significantly activated by the addition of dextran, but glucan yields were also lower in these mutants.

Production of natural antimicrobial compound D-phenyllactic acid using Leuconostoc mesenteroides ATCC 8293 whole cells involving highly active D-lactate dehydrogenase.

Phenyllactic acid (PLA) is an antimicrobial compound naturally synthesized in various fermented foods and its D-form of PLA is known to be more active than the L-isomer. In this study, Leuconostoc mesenteroides ATCC 8293 cells, elaborating D-lactate dehydrogenase (D-ldh) were used to produce D-PLA from phenylpyruvic acid (PPA). When cultured in the presence of PPA (≤50 mmol l(-1)), growing cells produced a maximum yield of 35 mmol l(-1) of D-PLA, and the yields were between 75·2 and 83·3%. Higher conversion yields were obtained at pH 6·0-7·0 when growing cells were used, while the optimum pH range was broader for resting cells. The time required for the complete conversion of PPA into PLA could be shortened to 3 h using resting cells. D-ldh, an enzyme encoded by the LEUM_1756 gene of Leuc. mesenteroides ATCC 8293, was found to be responsible for the conversion of PPA into PLA. The Km and kcat values of the enzyme for PPA were found to be 15·4 mmol l(-1) and 5645 s(-1), respectively. The conditions required for the efficient production of D-PLA were optimized for both growing and resting cells of Leuc. mesenteroides, with special emphasis on achieving high stereoselectivity and conversion yield. Significance and impact of the study: This is the first study on the production of D-phenyllactic acid, which is a natural antimicrobial compound, from phenylpyruvate using Leuconostoc mesenteroides cells. The strain, ATCC 8293, that was used in the study, possesses high stereoselectivity and delivers a high yield. Therefore, it might be a promising candidate for use in large-scale production facilities and in fermented foods.

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase.

ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN(123)-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.

Synthesis of 2,3-butanediol by Synechocystis sp. PCC6803 via heterologous expression of a catabolic pathway from lactic acid- and enterobacteria.

The direct and efficient conversion of CO2 into liquid energy carriers and/or bulk chemicals is crucial for a sustainable future of modern society. Here we describe the production of 2,3-butanediol in Synechocystis sp. PCC6803 expressing a heterologous catabolic pathway derived from enteric- and lactic acid bacteria. This pathway is composed of an acetolactate synthase, an acetolactate decarboxylase and an acetoin reductase. Levels of up to 0.72 g/l (corresponding to 8 mmol/L) of C(4) products, including a level of 0.43 g/l (corresponding to 4.7 mmol/L) 2,3-butanediol production are observed with the genes encoding these three enzymes integrated into the cyanobacterial genome, as well as when they are plasmid encoded. Further optimization studies revealed that Synechocystis expresses significant levels of acetolactate synthase endogenously, particularly under conditions of restricted CO2 supply to the cells. Co-expression of a soluble transhydrogenase or of an NADPH-dependent acetoin reductase allows one to drive the last step of the engineered pathway to near completion, resulting in pure meso-2,3-butanediol being produced.