•Lactate was selected as the target co-product produced with 1,3-propanediol.•By-product pathways were blocked in Klebsiella oxytoca PDL-0 for co-production.•Lactate production balanced glycerol metabolism and drove 1,3-propanediol production.•Over 70 g/L 1,3-propanediol and over 100 g/L optically pure lactate were produced.•Total conversion yields of target products highly reached over 0.95 mol/mol, respectively.Metabolic engineering has emerged as a powerful tool for bioproduction of both fine and bulk chemicals. The natural coordination among different metabolic pathways contributes to the complexity of metabolic modification, which hampers the development of biorefineries. Herein, the coordination between the oxidative and reductive branches of glycerol metabolism was rearranged in Klebsiella oxytoca to improve the 1,3-propanediol production. After deliberating on the product value, carbon conservation, redox balance, biological compatibility and downstream processing, the lactate-producing pathway was chosen for coupling with the 1,3-propanediol-producing pathway. Then, the other pathways of 2,3-butanediol, ethanol, acetate, and succinate were blocked in sequence, leading to improved d-lactate biosynthesis, which as return drove the 1,3-propanediol production. Meanwhile, efficient co-production of 1,3-propanediol and l-lactate was also achieved by replacing ldhD with ldhL from Bacillus coagulans. The engineered strains PDL-5 and PLL co-produced over 70 g/L 1,3-propanediol and over 100 g/L optically pure d-lactate and l-lactate, respectively, with high conversion yields of over 0.95 mol/mol from glycerol.

•A silent glycerol catabolism pathway was activated in Klebsiella pneumoniae.•The activation switched metabolic status and facilitated acetoin production.•High-level microbial production of acetoin was achieved from glycerol.•Sophisticated cross-talk was revealed between silent and active pathways.The physiological roles of silent genes are still unsolved puzzles and their application potentials are unexplored. Herein, a silent glycerol catabolism pathway encoded by glp system was activated in a Klebsiella pneumoniae mutant, of which the acetoin degradation pathway was blocked. Surprisingly, the activation produced significant effects on cellular metabolism and over 90% of the carbon flux was redirected to acetoin biosynthesis, which was formerly a minor product in this mutant. Transcription analyses suggest that the genes involved in acetoin, 1,3-propanediol and adenosyl-cobalamin biosynthesis were differentially regulated upon the glp system activation, demonstrating the cross-talk between silent and active pathways. Through pathway, cofactor and bioprocess engineering, high-level acetoin production from glycerol (32.2 g L−1, 90.8% of the theoretical value) was achieved. Our findings suggest that some silent genes represent unexplored switches of cellular metabolic status and activating them may be an easy strategy for reprogramming microorganisms into efficient cell factories.

Many plant natural products have remarkable pharmacological activities. They are mainly produced directly by extraction from higher plants, which can hardly keep up with the surging global demand. Furthermore, the over-felling of many medicinal plants has undesirable effects on the ecological balance. In this study, we constructed a photoautotrophic platform with the unicellular cyanobacterium Synechococcus elongatus PCC7942 to directly convert the greenhouse gas CO2 into an array of valuable healthcare products, including resveratrol, naringenin, bisdemethoxycurcumin, p-coumaric acid, caffeic acid, and ferulic acid. These six compounds can be further branched to many other precious and useful natural products. Various strategies including introducing a feedback-inhibition-resistant enzyme, creating functional fusion proteins, and increasing malonyl-CoA supply have been systematically investigated to increase the production. The highest titers of these natural products reached 4.1–128.2 mg L−1 from the photoautotrophic system, which are highly comparable with those obtained by many other heterotrophic microorganisms using carbohydrates. Several advantages such as independence from carbohydrate feedstocks, functionally assembling P450s, and availability of plentiful NADPH and ATP support that this photosynthetic platform is uniquely suited for producing plant natural products. This platform also provides a green route for direct conversion of CO2 to many aromatic building blocks, a promising alternative to petrochemical-based production of bulk aromatic compounds.

Platform chemicals can be readily converted into various value-added chemicals and fuels. Photosynthetic production of platform chemicals directly from CO2 by cyanobacteria, in the presence of sunlight, holds promise for addressing global energy and environmental concerns. Herein, we report the photosynthetic production of C3 platform chemicals using engineered Synechococcus elongatus PCC7942 as the kernel. The engineered S. elongatus strain YW1 expressing glycerol-3-phosphatase produced a C3 intermediate, glycerol, with a high concentration of 1.17 g L−1 and a maximum production rate of 7733 μg L−1 H−1. Strain YW1 could serve as the kernel for the production of various C3 chemicals. By extending heterologous pathways in the cyanobacterial kernel, the carbon flux was further channelled to produce two platform chemicals: dihydroxyacetone by introducing glycerol dehydrogenase and 3-hydroxypropionic acid by introducing glycerol dehydratase and aldehyde dehydrogenase. Co-cultivation of the cyanobacterial kernel and another microbe, Klebsiella pneumoniae, was also performed to convert the C3 intermediate produced from CO2 to 1,3-propanediol, an important monomer for biodegradable material production. Besides direct photosynthetic production and co-cultivation, we demonstrated that glycerol produced by the cyanobacterial kernel can be used as a fermentation feedstock after simple concentration. The production processes presented here display great potential for carbon capture and storage and for sustainable production of chemicals and fuels.

Conversion of glycerol into high-value products is of significant importance for sustainability in the biofuel industry. In this study, pyruvic acid, a central intermediate needed for the production of versatile biomolecules, was produced from glycerol without the addition of any cofactors by the cell-free bio-system composed of alditol oxidase, dihydroxy acid dehydratase, and catalase. (3R)-Acetoin was then produced at 85.5% of the theoretical yield from glycerol by α-acetolactate synthase and α-acetolactate decarboxylase. Since other biomolecules can also be produced from pyruvic acid, the cell-free bio-system might serve as a versatile bio-production platform, and support the viability of the biofuel economy.

The twin-arginine translocation (Tat) pathway exports folded proteins across the cytoplasmic membranes of bacteria and archaea. Two parallel Tat pathways (TatAdCd and TatAyCy systems) with distinct substrate specificities have previously been discovered in Bacillus subtilis. In this study, to secrete methyl parathion hydrolase (MPH) into the growth medium, the twin-arginine signal peptide of B. subtilis YwbN was used to target MPH to the Tat pathway of B. subtilis. Western blot analysis and MPH assays demonstrated that active MPH was secreted into the culture supernatant of wild-type cells. No MPH secretion occurred in a total-tat2 mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. Export was fully blocked in a tatAyCy mutant. In contrast, the tatAdCd mutant was still capable of secreting MPH. These results indicated that the MPH secretion directed by the YwbN signal peptide was specifically mediated by the TatAyCy system. The N-terminal sequence of secreted MPH was determined as AAPQVR, demonstrating that the YwbN signal peptide had been processed correctly. This is the first report of functional secretion of a heterologous protein via the B. subtilis TatAyCy system. This study highlights the potential of the TatAyCy system to be used for secretion of other heterologous proteins in B. subtilis.

This paper reports the codisplay of organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH)–green fluorescent protein (GFP) fusion on the cell surface of Escherichia coli using the truncated ice nucleation protein (INPNC) and Lpp–OmpA as the anchoring motifs. The surface localization of both OPH and MPH–GFP was demonstrated by cell fractionation, Western blot analysis, protease accessibility experiment, and immunofluorescence microscopy. Anchorage of the foreign proteins on the outer membrane neither inhibits cell growth nor affects cell viability. The recombinant strain can be used as a whole-cell biocatalyst and showed a broader substrate range than strains expressing either OPH or MPH. A mixture of six organophosphorus pesticides (OPs) (0.2 mM each) could be degraded completely within 5 h. The broader substrate specificity in combination with the rapid degradation rate makes the recombinant strain a promising candidate for detoxification of OPs. The fluorescence of surface-displayed GFP is very sensitive to environmental pH change. Because hydrolysis of OPs by OPH or MPH generates protons, the recombinant E. coli could be used as a whole-cell biosensor for the rapid detection of OPs by evaluating fluorescence changes as a function of OP concentrations.

Highly efficient d-lactate production by Sporolactobacillus sp. strain CASD was demonstrated in this study. Peanut meal was found to be a better nutrient than yeast extract, soybean meal, soybean peptone, corn steep, liquor beef extract, and ammonium sulfate in the production of d-lactate. To improve the utilization of peanut meal, the material was enzymatically hydrolyzed and simultaneously utilized as the nitrogen source in d-lactate fermentation. Very high d-lactate production (207 g/L) was obtained using 40 g/L of peanut meal in 30-L fed-batch fermentation, with the average productivity of 3.8 g/(L·h) and optical purity of 99.3%. The production of such a high concentration of optically pure d-lactate by strain CASD, with the simultaneous enzymatic hydrolysis of peanut meal and fermentation, represents a new cost-efficient and integrated method for d-lactate production using agricultural by-products.

Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet™-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.

N-Acetyl-d-neuraminic acid (Neu5Ac) and its derivates are a very important group of biomolecules because these sugars occupy the terminal positions in numerous macromolecules, such as the glycans of glycoproteins, and are involved in many biological and pathological phenomena. The synthesis and applications of Neu5Ac are attracting much interest due to the potential applications of this compound in the pharmaceutical industry, such as in the synthesis of the anti-flu drug zanamivir. In this review article, we discuss existing knowledge on the biotechnological production and applications of Neu5Ac and also propose some guidelines for future studies.

N-Acetyl-d-neuraminic acid (Neu5Ac) is a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1. Two recombinant Escherichia coli strains capable of expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid aldolase were constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in Neu5Ac production. Carbon sources were optimized for Neu5Ac production, and the concentration effects of carbon sources on the production were investigated. With 2,200 mM pyruvate as carbon source and substrate, 61.9 mM (19.1 g l−1) Neu5Ac was produced from 200 mM N-acetyl-d-glucosamine (GlcNAc) in 36 h by the coupled cells. Our Neu5Ac biosynthetic process is favorable compared with natural product extraction, chemical synthesis, or even many other biocatalysis processes.

A simple consortium consisted of two members of Klebsiella sp. A1 and Comamonas sp. A2 was isolated from the sewage of a pesticide mill in China. One member of Klebsiella sp. A1 is a novel strain that could use atrazine as the sole carbon and nitrogen source. The consortium showed high atrazine-mineralizing efficiency and about 83.3% of 5 g l−1 atrazine could be mineralized after 24 h degradation. Contrary to many other reported microorganisms, the consortium was insensitive to some nitrogenous fertilizers commonly used, not only in presence of 200 mg l−1 atrazine but also in 5 g l−1 atrazine mediums. After 24 h incubation, 200 mg l−1 atrazine was completely mineralized despite of the presence of urea, (NH4)2CO3 and (NH4)2HPO4 in the medium. Very minor influence was observed when NH4Cl was added as additional nitrogen source. Advantages of the simple consortium, high mineralizing efficiency and insensitivity to most of exogenous nitrogen sources, all suggested application potential of the consortium for the bioremediation of atrazine-contaminated soils and waters.

A biphenyl (BP)-utilizing bacterium, designated B6-2, was isolated from soil and identified as Pseudomonas putida. BP-grown B6-2 cells were capable of transforming dibenzofuran (DBF) via a lateral dioxygenation and meta-cleavage pathway. The ring cleavage product 2-hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid (HOBB) was detected as a major metabolite. B6-2 growing cells could also cometabolically degrade DBF using BP as a primary substrate. A recombinant Escherichia coli strain DH10B (pUC118bphABC) expressing BP dioxygenase, BP-dihydrodiol dehydrogenase, and dihydroxybiphenyl dioxygenase was shown to be capable of transforming DBF to HOBB. Using purified HOBB that was produced by the recombinant as the substrate for B6-2, we newly identified a series of benzofuran derivatives as metabolites. The structures of these metabolites indicate that an unreported HOBB degradation pathway is employed by strain B6-2. In this pathway, HOBB is proposed to be transformed to 2-oxo-4-(3′-oxobenzofuran-2′-yl)butanoic acid and 2-hydroxy-4-(3′-oxobenzofuran-2′-yl)butanoic acid (D4) through two sequential double-bond hydrogenation steps. D4 is suggested to undergo reactions including decarboxylation and oxidation to produce 3-(3′-oxobenzofuran-2′-yl)propanoic acid (D6). 3-Hydroxy-3-(3′-oxobenzofuran-2′-yl)propanoic acid (D7) and 2-(3′-oxobenzofuran-2′-yl)acetic acid (D8) would represent metabolites involved in the processes of beta- and alpha-oxidation of D6, respectively. D7 and D8 are suggested to be transformed to their respective products 3-hydroxy-2,3-dihydrobenzofuran-2-carboxylic acid (D10) and 2-(3′-hydroxy-2′,3′-dihydrobenzofuran-2′-yl)acetic acid. D10 is proposed to be transformed to salicylic acid (D14) via 2,3-dihydro-2,3-dihydroxybenzofuran, 2-oxo-2-(2′-hydroxyphenyl)acetic acid and 2-hydroxy-2-(2′-hydroxyphenyl)acetic acid. Further experimental results revealed that B6-2 was capable of growing with D14 as the sole carbon source. Because benzofuran derivatives may have biological, pharmacological, and toxic properties, the elucidation of this new pathway should be significant from both biotechnological and environmental views.

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH4)2HPO4 were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation.

•We provide a categorization of NAD-independent lactate dehydrogenases (iLDHs).•We introduce how iLDHs are applied in industry and human life.•We summarize how lactate utilization stimulates microbial pathogenicity.•We compare the regulation mechanisms of lactate utilization in different species.Lactate utilization endows microbes with the ability to use lactate as a carbon source. Lactate oxidizing enzymes play key roles in the lactate utilization pathway. Various types of these enzymes have been characterized, but novel ones remain to be identified. Lactate determination techniques and biocatalysts have been developed based on these enzymes. Lactate utilization has also been found to induce pathogenicity of several microbes, and the mechanisms have been investigated. More recently, studies on the structure and organization of operons of lactate utilization have been carried out. This review focuses on the recent progress and future perspectives in understanding microbial lactate utilization.

Muconic acid (MA), a high value-added bio-product with reactive dicarboxylic groups and conjugated double bonds, has garnered increasing interest owing to its potential applications in the manufacture of new functional resins, bio-plastics, food additives, agrochemicals, and pharmaceuticals. At the very least, MA can be used to produce commercially important bulk chemicals such as adipic acid, terephthalic acid and trimellitic acid. Recently, great progress has been made in the development of biotechnological routes for MA production. This present review provides a comprehensive and systematic overview of recent advances and challenges in biotechnological production of MA. Various biological methods are summarized and compared, and their constraints and possible solutions are also described. Finally, the future prospects are discussed with respect to the current state, challenges, and trends in this field, and the guidelines to develop high-performance microbial cell factories are also proposed for the MA production by systems metabolic engineering.

•Mycobacterium goodii X7B has the ability to remove organic sulfur from a broad range of sulfur species in gasoline, diesel and crude oil.•The predominant properties make M. goodii X7B as a potential workhorse for petroleum biodesulfurization process.•The first released genome sequence of facultative thermophilic biodesulfurizing strain M. goodii X7B may facilitate the further strain improvements to increase the efficiency.Mycobacterium goodii X7B appeared to have the ability to remove organic sulfur from a broad range of sulfur species in gasoline, diesel and crude oils. The predominant properties make it as a potential workhorse for petroleum biodesulfurization process. We sequenced and annotated the whole genome to serve as a basis for further elucidation of the genetic background of this promising strain, and provide opportunities for investigating the metabolic and regulatory mechanisms.

Acidogenic and aciduric bacteria have developed several survival systems in various acidic environments to prevent cell damage due to acid stress such as that on the human gastric surface and in the fermentation medium used for industrial production of acidic products. Common mechanisms for acid resistance in bacteria are proton pumping by F1–F0–ATPase, the glutamate decarboxylase system, formation of a protective cloud of ammonia, high cytoplasmic urease activity, repair or protection of macromolecules, and biofilm formation. The field of synthetic biology has rapidly advanced and generated an ever-increasing assortment of genetic devices and biological modules for applications in biofuel and novel biomaterial productions. Better understanding of aspects such as overproduction of general shock proteins, molecular mechanisms, and responses to cell density adopted by microorganisms for survival in low pH conditions will prove useful in synthetic biology for potential industrial and environmental applications.