Engineering the Outer Membrane Could Facilitate Better Bacterial Performance and Effectively Enhance Poly-3-Hydroxybutyrate Accumulation.

Poly-3-hydroxybutyrate (PHB) is an environmentally friendly polymer and can be produced in Escherichia coli cells after overexpression of the heterologous gene cluster phaCAB. The biosynthesis of the outer membrane (OM) consumes many nutrients and influences cell morphology. Here, we engineered the OM by disrupting all gene clusters relevant to the polysaccharide portion of lipopolysaccharide (LPS), colanic acid (CA), flagella, and/or fimbria in E. coli W3110. All these disruptions benefited PHB production. Especially, disrupting all these OM components increased the PHB content to 83.0 wt% (PHB content percentage of dry cell weight), while the wild-type control produced only 1.5 wt% PHB. The increase was mainly due to the LPS truncation to Kdo2 (3-deoxy-d-manno-octulosonic acid)-lipid A, which resulted in 82.0 wt% PHB with a 25-fold larger cell volume, and disrupting CA resulted in 57.8 wt% PHB. In addition, disrupting LPS facilitated advantageous fermentation features, including 69.1% less acetate, a 550% higher percentage of autoaggregated cells among the total culture cells, 69.1% less biofilm, and a higher broken cell ratio. Further detailed mechanism investigations showed that disrupting LPS caused global changes in envelope and cellular metabolism: (i) a sharp decrease in flagella, fimbria, and secretions; (ii) more elastic cells; (iii) much greater carbon flux toward acetyl coenzyme A (acetyl-CoA) and supply of cofactors, including NADP, NAD, and ATP; and (iv) a decrease in by-product acids but increase in γ-aminobutyric acid by activating σE factor. Disrupting CA, flagella, and fimbria also improved the levels of acetyl-CoA and cofactors. The results indicate that engineering the OM is an effective strategy to enhance PHB production and highlight the applicability of OM engineering to increase microbial cell factory performance. IMPORTANCE Understanding the detailed influence of the OM on the cell envelope and cellular metabolism is important for optimizing the E. coli cell factory and many other microorganisms. This study revealed the applicability of remodeling the OM to enhance PHB accumulation as representative inclusion bodies. The results generated in this study give essential information for producing other inclusion bodies or chemicals which need more acetyl-CoA and cofactors but less by-product acids. This study is promising to provide new ideas for the improvement of microbial cell factories.

Engineering Clostridium cellulovorans for highly selective n-butanol production from cellulose in consolidated bioprocessing.

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlACA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbdCT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P)+ oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnrCA -thlACA -hbdCT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biopolymer by recombinant Bacillus megaterium in fed-batch bioreactors.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated in a wide variety of microorganisms as intracellular carbon and energy storage compounds. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the most valuable biopolymers because of its superior mechanical properties. Here, we developed a bioprocess utilizing recombinant Bacillus megaterium strain for PHBV over-production from glucose, without any precursor addition. PHA production was performed in a controlled bioreactor by batch and fed-batch modes using wild-type B. megaterium and rec-B. megaterium cells overexpressing the native phaC gene. The effect of oxygen transfer rate on biomass formation and PHA accumulation was also investigated, under different dissolved oxygen levels. Structural and thermal properties of PHA were characterized by GC-FID, 1H-NMR, TGA and DSC analyses. Significantly, the copolymer produced from glucose as the carbon source in rec-B. megaterium was composed of 58 mol% of 3-hydroxyvalerate monomers. After 66 h, rec-B. megaterium cells in fed-batch fermentation with a pre-determined growth rate µ0 = 0.1 h-1 produced the highest CDW (7.7 g L-1) and PHA concentration (6.1 g L-1). Moreover, an exponential glucose feeding profile resulted in 2.2-fold increase in PHA yield compared to batch cultivation. Overall, this study paves the way to an enhanced biopolymer production process in B. megaterium cells, where the highest product yield on cell was obtained as YP/X = 0.8 g g-1.

Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters.

As concerns increase regarding sustainable industries and environmental pollutions caused by the accumulation of non-degradable plastic wastes, bio-based polymers, particularly biodegradable plastics, have attracted considerable attention as potential candidates for solving these problems by substituting petroleum-based plastics. Among these candidates, polyhydroxyalkanoates (PHAs), natural polyesters that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and material properties similar to those of commodity plastics. Accordingly, substantial efforts have been made to gain a better understanding of mechanisms related to the biosynthesis and properties of PHAs and to develop natural and recombinant microorganisms that can efficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent advances in biotechnology, including those related to evolutionary engineering, synthetic biology, and systems biology, can provide efficient and effective tools and strategies that reduce time, labor, and costs to develop microbial platform strains that produce desired chemicals and materials. Adopting these technologies in a systematic manner has enabled microbial fermentative production of non-natural polyesters such as poly(lactate) [PLA], poly(lactate-co-glycolate) [PLGA], and even polyesters consisting of aromatic monomers from renewable biomass-derived carbohydrates, which can be widely used in current chemical industries. In this review, we present an overview of strain development for the production of various important natural PHAs, which will give the reader an insight into the recent advances and provide indicators for the future direction of engineering microorganisms as plastic cell factories. On the basis of our current understanding of PHA biosynthesis systems, we discuss recent advances in the approaches adopted for strain development in the production of non-natural polyesters, notably 2-hydroxycarboxylic acid-containing polymers, with particular reference to systems metabolic engineering strategies.

Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application.

Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as "melanin ghost" structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis.IMPORTANCE Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens-produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.

Proteome analysis guided genetic engineering of Corynebacterium glutamicum S9114 for tween 40-triggered improvement in L-ornithine production.

BACKGROUND: L-ornithine is a valuable amino acid with a wide range of applications in the pharmaceutical and food industries. However, the production of L-ornithine by fermentation cannot compete with other methods, because of the low titers produced with this technique. Development of fermentation techniques that result in a high yield of L-ornithine and efficient strategies for improving L-ornithine production are essential. RESULTS: This study demonstrates that tween 40, a surfactant promoter of the production of glutamate and arginine, improves L-ornithine production titers in engineered C. glutamicum S9114. The intracellular metabolism under tween 40 triggered fermentation conditions was explored using a quantitative proteomic approach, identifying 48 up-regulated and 132 down-regulated proteins when compared with the control. Numerous proteins were identified as membrane proteins or functional proteins involved in the biosynthesis of the cell wall. Modulation of those genes revealed that the overexpression of CgS9114_09558 and the deletion of CgS9114_13845, CgS9114_02593, and CgS9114_02058 improved the production of L-ornithine in the engineered strain of C. glutamicum Orn8. The final strain with all the exploratory metabolic engineering manipulations produced 25.46 g/L of L-ornithine, and a yield of 0.303 g L-ornithine per g glucose, which was 30.6% higher than that produced by the original strain (19.5 g/L). CONCLUSION: These results clearly demonstrate the positive effect of tween 40 addition on L-ornithine accumulation. Proteome analysis was performed to examine the impact of tween 40 addition on the physiological changes in C. glutamicum Orn8 and the results showed several promising modulation targets for developing L-ornithine-producing strains.

Engineering the cellulolytic extreme thermophile Caldicellulosiruptor bescii to reduce carboxylic acids to alcohols using plant biomass as the energy source.

Caldicellulosiruptor bescii is the most thermophilic cellulolytic organism yet identified (Topt 78 °C). It grows on untreated plant biomass and has an established genetic system thereby making it a promising microbial platform for lignocellulose conversion to bio-products. Here, we investigated the ability of engineered C. bescii to generate alcohols from carboxylic acids. Expression of aldehyde ferredoxin oxidoreductase (aor from Pyrococcus furiosus) and alcohol dehydrogenase (adhA from Thermoanaerobacter sp. X514) enabled C. bescii to generate ethanol from crystalline cellulose and from biomass by reducing the acetate produced by fermentation. Deletion of lactate dehydrogenase in a strain expressing the AOR-Adh pathway increased ethanol production. Engineered strains also converted exogenously supplied organic acids (isobutyrate and n-caproate) to the corresponding alcohol (isobutanol and hexanol) using both crystalline cellulose and switchgrass as sources of reductant for alcohol production. This is the first instance of an acid to alcohol conversion pathway in a cellulolytic microbe.

Perspectives and new directions for bioprocess optimization using Zymomonas mobilis in the ethanol production.

Zymomonas mobilis is an ethanologenic microbe that has a demonstrated potential for use in lignocellulosic biorefineries for bioethanol production. Z. mobilis exhibits a number of desirable characteristics for use as an ethanologenic microbe, with capabilities for metabolic engineering and bioprocess modification. Many advanced genetic tools, including mutation techniques, screening methods and genome editing have been successively performed to improve various Z. mobilis strains as potential consolidated ethanologenic microbes. Many bioprocess strategies have also been applied to this organism for bioethanol production. Z. mobilis biofilm reactors have been modified with various benefits, including high bacterial populations, less fermentation times, high productivity, high cell stability, resistance to the high concentration of substrates and toxicity, and higher product recovery. We suggest that Z. mobilis biofilm reactors could be used in bioethanol production using lignocellulosic substrates under batch, continuous and repeated batch processes.

Metabolically engineered Lactobacillus gasseri JCM 1131 as a novel producer of optically pure L- and D-lactate.

High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with poly-D-lactate. On an industrial scale, this process simultaneously consumes large amounts of both optically pure lactate stereoisomers. However, because optimal growth conditions of L-lactate producers often differ from those of D-lactate producers, each stereoisomer is produced in a specialised facility, which raises cost and lowers sustainability. To address this challenge, we metabolically engineered Lactobacillus gasseri JCM 1131T, a bioprocess-friendly and genetically malleable strain of homofermentative lactic acid bacterium, to efficiently produce either pure L- or pure D-lactate under the same bioprocess conditions. Transformation of L. gasseri with plasmids carrying additional genes for L- or D-lactate dehydrogenases failed to affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96 g of either L- or D-lactate per gram of glucose. In this study, the plasmid pHBintE, routinely used for gene disruption in Bacillus megaterium, was used for the first time to inactivate genes in lactobacilli. Strains with inactivated genes for endogenous lactate dehydrogenases efficiently fermented sugars released by enzymatic hydrolysis of alkali pre-treated wheat straw, an abundant lignocellulose-containing raw material, producing 0.37-0.42 g of lactate per gram of solid part of alkali-treated wheat straw. Thus, the constructed strains are primed to serve as producers of both optically pure L-lactate and D-lactate in the next-generation biorefineries.

Improved n-Butanol Production from Clostridium cellulovorans by Integrated Metabolic and Evolutionary Engineering.

Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing adhE1 and ctfA-ctfB-adc genes from Clostridium acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward n-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.IMPORTANCE Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n-butanol production from lignocellulose by CBP.

Redesigning the Aspergillus nidulans xylanase regulatory pathway to enhance cellulase production with xylose as the carbon and inducer source.

BACKGROUND: Biomass contains cellulose (C6-sugars), hemicellulose (C5-sugars) and lignin. Biomass ranks amongst the most abundant hydrocarbon resources on earth. However, biomass is recalcitrant to enzymatic digestion by cellulases. Physicochemical pretreatment methods make cellulose accessible but partially destroy hemicellulose, producing a C5-sugar-rich liquor. Typically, digestion of pretreated LCB is performed with commercial cellulase preparations, but C5-sugars could in principle be used for "on site" production of cellulases by genetically engineered microorganism, thereby reducing costs. RESULTS: Here we report a succession of genetic interventions in Aspergillus nidulans that redesign the natural regulatory circuitry of cellulase genes in such a way that recombinant strains use C5-sugar liquors (xylose) to grow a vegetative tissue and simultaneously accumulate large amounts of cellulases. Overexpression of XlnR showed that under xylose-induction conditions only xylanase C was produced. XlnR overexpression strains were constructed that use the xynCp promoter to drive the production of cellobiohydrolases, endoglucanases and ß-glucosidase. All five cellulases accumulated at high levels when grown on xylose. Production of cellulases in the presence of pretreated-biomass C5-sugar liquors was investigated, and cellulases accumulated to much higher enzyme titers than those obtained for traditional fungal cell factories with cellulase-inducing substrates. CONCLUSIONS: By replacing expensive substrates with a cheap by-product carbon source, the use of C5-sugar liquors directly derived from LCB pretreatment processes not only reduces enzyme production costs, but also lowers operational costs by eliminating the need for off-site enzyme production, purification, concentration, transport and dilution.

Engineering Clostridium for improved solvent production: recent progress and perspective.

Clostridia are Gram-positive, spore-forming, obligate anaerobic bacteria that can produce solvents such as acetone, ethanol, and butanol, which can be used as biofuels or building block chemicals. Many successful attempts have been made to improve solvent yield and titer from sugars through metabolic engineering of solventogenic and acidogenic clostridia. More recently, cellulolytic and acetogenic clostridia have also attracted high interests for their ability to utilize low-cost renewable substrates such as cellulose and syngas. Process engineering such as in situ butanol recovery and consolidated bioprocessing (CBP) has been developed for improved solvent titer and productivity. This review focuses on metabolic and process engineering strategies for solvent production from sugars, lignocellulosic biomass, and syngas by various clostridia, including conventional solventogenic Clostridium acetobutylicum, engineered acidogens such as C. tyrobutyricum and C. cellulovorans, and carboxydotrophic acetogens such as C. carboxidivorans and C. ljungdahlii.

Bioconversion of lignin and its derivatives into polyhydroxyalkanoates: Challenges and opportunities.

Renewable energy resources are considered to be promising for the development of a sustainable circular economy. Among various alternatives, the microbial route for various biofuels production is quite lucrative. Use of cellulose and lignocellulose for methane, H2 , organic acids, ethanol, and cellulase has been explored a lot in the past few decades. The major leftover or a coproduct of these processes belongs to lignin-an aromatic cross-link polymer and one of the most abundant complex compounds on earth. A successful bioconversion route of lignin into high-value products is highly desirable for biorefinery perspective. It requires a complex set of enzymes/catalysts to decompose lignin through depolymerization and oxygen removal leading to its monomers that can be metabolized by engineered organisms to synthesize muconic acids, polyhydroxyalkanoates (PHAs), methane, and other high-value products. This article will focus on the opportunities and challenges in the bioconversion of lignin and its derivatives into PHAs.

From lignin to nylon: Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida.

Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L-1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L-1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.

Investigating the strategies for microbial production of trehalose from lignocellulosic sugars.

Trehalose, a multi-functional and value-added disaccharide, can be efficiently biosynthesized from glucose by using a synergetic carbon utilization mechanism (SynCar) which coupled phosphoenolpyruvate (PEP) generation from the second carbon source with PEP-dependent phosphotransferase system (PTS) to promote non-catabolic use of glucose. Considering glucose and xylose present in large amounts in lignocellulosic sugars, we explored new strategies for conversion of both sugars into trehalose. Herein, we first attempted trehalose production from xylose directly, based on which, synergetic utilization of glucose, and xylose prompted by SynCar was implemented in engineered Escherichia coli. As the results, the final titer of trehalose reached 5.55 g/L in shake flask experiments. The conversion ratio or utilization efficiency of glucose or xylose to trehalose was around fourfold higher than that of the original strain (YW-3). This work not only demonstrated the possibility of directly converting xylose (C5 sugar) into trehalose (C12 disaccharide), but also suggested a promising strategy for trehalose production from lignocellulosic sugars for the first time.

L-Lactic acid production from glucose and xylose with engineered strains of Saccharomyces cerevisiae: aeration and carbon source influence yields and productivities.

BACKGROUND: Saccharomyces cerevisiae, engineered for L-lactic acid production from glucose and xylose, is a promising production host for lignocellulose-to-lactic acid processes. However, the two principal engineering strategies-pyruvate-to-lactic acid conversion with and without disruption of the competing pyruvate-to-ethanol pathway-have not yet resulted in strains that combine high lactic acid yields (YLA) and productivities (QLA) on both sugar substrates. Limitations seemingly arise from a dependency on the carbon source and the aeration conditions, but the underlying effects are poorly understood. We have recently presented two xylose-to-lactic acid converting strains, IBB14LA1 and IBB14LA1_5, which have the L-lactic acid dehydrogenase from Plasmodium falciparum (pfLDH) integrated at the pdc1 (pyruvate decarboxylase) locus. IBB14LA1_5 additionally has its pdc5 gene knocked out. In this study, the influence of carbon source and oxygen on YLA and QLA in IBB14LA1 and IBB14LA1_5 was investigated. RESULTS: In anaerobic fermentation IBB14LA1 showed a higher YLA on xylose (0.27 g g Xyl-1 ) than on glucose (0.18 g g Glc-1 ). The ethanol yields (YEtOH, 0.15 g g Xyl-1 and 0.32 g g Glc-1 ) followed an opposite trend. In IBB14LA1_5, the effect of the carbon source on YLA was less pronounced (~ 0.80 g g Xyl-1 , and 0.67 g g Glc-1 ). Supply of oxygen accelerated glucose conversions significantly in IBB14LA1 (QLA from 0.38 to 0.81 g L-1 h-1) and IBB14LA1_5 (QLA from 0.05 to 1.77 g L-1 h-1) at constant YLA (IBB14LA1 ~ 0.18 g g Glc-1 ; IBB14LA1_5 ~ 0.68 g g Glc-1 ). In aerobic xylose conversions, however, lactic acid production ceased completely in IBB14LA1 and decreased drastically in IBB14LA1_5 (YLA aerobic ≤ 0.25 g g Xyl-1 and anaerobic ~ 0.80 g g Xyl-1 ) at similar QLA (~ 0.04 g L-1 h-1). Switching from aerobic to microaerophilic conditions (pO2 ~ 2%) prevented lactic acid metabolization, observed for fully aerobic conditions, and increased QLA and YLA up to 0.11 g L-1 h-1 and 0.38 g g Xyl-1 , respectively. The pfLDH and PDC activities in IBB14LA1 were measured and shown to change drastically dependent on carbon source and oxygen. CONCLUSION: Evidence from conversion time courses together with results of activity measurements for pfLDH and PDC show that in IBB14LA1 the distribution of fluxes at the pyruvate branching point is carbon source and oxygen dependent. Comparison of the performance of strain IBB14LA1 and IBB14LA1_5 in conversions under different aeration conditions (aerobic, anaerobic, and microaerophilic) further suggest that xylose, unlike glucose, does not repress the respiratory response in both strains. This study proposes new genetic engineering targets for rendering genetically engineering S. cerevisiae better suited for lactic acid biorefineries.

Production of Bioplastic Compounds by Genetically Manipulated and Metabolic Engineered Cyanobacteria.

Direct conversion of carbon dioxide to valuable compounds is a desirable way to reduce the environmental burden and switch from fossil to renewable fuels. Cyanobacteria are photosynthetic bacteria that perform oxygenic photosynthesis and are able to produce valuable compounds from carbon dioxide in the air. Synechocystis and Synechococcus species, model unicellular cyanobacteria, can produce succinate and lactate, which are commodity chemicals used to generate bioplastics. Several cyanobacteria are also able to produce polyhydroxybutyrate, a biodegradable polyester that accumulates under nitrogen or phosphorus starvation. Genetic manipulation succeeded in increasing the productivity of succinate, lactate, and polyhydroxybutyrate from cyanobacteria. We summarize the recent findings in this review.

Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli.

Bacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccp Ax). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 °C, 30 °C, and 37 °C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000-3000 µm and a diameter of 10-20 µm, which were remarkably larger than the fibres of BC typically produced by G. hansenii.

Biosynthesis of Natural Rubber: Current State and Perspectives.

Natural rubber is a kind of indispensable biopolymers with great use and strategic importance in human society. However, its production relies almost exclusively on rubber-producing plants Hevea brasiliensis, which have high requirements for growth conditions, and the mechanism of natural rubber biosynthesis remains largely unknown. In the past two decades, details of the rubber chain polymerization and proteins involved in natural rubber biosynthesis have been investigated intensively. Meanwhile, omics and other advanced biotechnologies bring new insight into rubber production and development of new rubber-producing plants. This review summarizes the achievements of the past two decades in understanding the biosynthesis of natural rubber, especially the massive information obtained from the omics analyses. Possibilities of natural rubber biosynthesis in vitro or in genetically engineered microorganisms are also discussed.

Consolidated bioprocessing for cellulosic ethanol conversion by cellulase-xylanase cell-surfaced yeast consortium.

Consolidated bioprocessing (CBP) strategy was developed to construct a cell-surface displayed consortium for heterologously expressing functional lignocellulytic enzymes. The reaction system composed of two engineered yeast strains: Y5/XynII-XylA (co-displaying two types of xylanases) and Y5/EG-CBH-BGL (co-displaying three types of cellulases). The immobilization of recombinant fusion proteins and their cell-surface accessibility of were analyzed by flow cytometry and immunofluorescence. The feasibility of consolidated bioprocessing by using pretreated corn stover (CS) as substrate for direct bioconversion was further investigated, and the synergistic activity and proximity effect between cellulases and xylanases on lignocelluloses degradation were also discussed in this work. Without any commercial enzyme addition, the combined yeast consortium produced 1.61 g/L ethanol which achieved 64.7% of the theoretical ethanol yield during 144 h from steam-exploded CS. The results indicated that the assembly of cellulases and xylanases using a synthetic consortium capable of combined displaying lignocellulytic enzymes is a promising approach for simultaneous saccharification and fermentation to ethanol from lignocellulosic biomass.