Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by Bacillus subtilis

Previously we constructed a Bacillus subtilis strain for efficient production of N-acetylglucosamine (GlcNAc) by engineering of GlcNAc synthetic and catabolic pathways. However, the further improvement of GlcNAc titer is limited by the intrinsic inefficiency of GlcNAc synthetic pathway and undesirable cellular properties including sporulation and high maintenance metabolism. In this work, we further improved GlcNAc titer through spatial modulation of key pathway enzymes and by blocking sporulation and decreasing maintenance metabolism. Specifically, a DNA-guided scaffold system was firstly used to modulate the activities of glucosamine-6-phosphate synthase and GlcNAc-6-phosphate N-acetyltransferase, increasing the GlcNAc titer from 1.83 g/L to 4.55 g/L in a shake flask. Next, sporulation was blocked by respectively deleting spo0A (gene encoding the initiation regulon of sporulation) and sigE (gene encoding RNA polymerase sporulation-specific sigma factor). Deletion of sigE more effectively blocked sporulation without altering cell growth or GlcNAc production. The respiration chain was then engineered to decrease the maintenance metabolism of recombinant B. subtilis by deleting cydB and cydC, genes encoding cytochrome bd ubiquinol oxidase (subunit II) and ATP-binding protein for the expression of cytochrome bd, respectively. The respiration-engineered B. subtilis produced 6.15 g/L GlcNAc in a shake flask and 20.58 g/L GlcNAc in a 3-L fed-batch bioreactor. To the best of our knowledge, this report is the first to describe the modulation of pathway enzymes via a DNA-guided scaffold system in B. subtilis. The combination of spatial modulation of key pathway enzymes and optimization of cellular properties may be used to develop B. subtilis as a well-organized cell factory for the production of the other industrially useful chemicals.     

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.     

Development of Halomonas TD01 as a host for open production of chemicals

Genetic engineering of Halomonas spp. was seldom reported due to the difficulty of genetic manipulation and lack of molecular biology tools. Halomonas TD01 can grow in a continuous and unsterile process without other microbial contaminations. It can be therefore exploited for economic production of chemicals. Here, Halomonas TD01 was metabolically engineered using the gene knockout procedure based on markerless gene replacement stimulated by double-strand breaks in the chromosome. When gene encoding 2-methylcitrate synthase in Halomonas TD01 was deleted, the conversion efficiency of propionic acid to 3-hydroxyvalerate (3HV) monomer fraction in random PHBV copolymers of 3-hydroxybutyrate (3HB) and 3HV was increased from around 10% to almost 100%, as a result, cells were grown to accumulate 70% PHBV in dry weight (CDW) consisting of 12 mol% 3HV from 0.5 g/L propionic acid in glucose mineral medium. Furthermore, successful deletions on three PHA depolymerases eliminate the possible influence of PHA depolymerases on PHA degradation in the complicated industrial fermentation process even though significant enhanced PHA content was not observed. In two 500 L pilot-scale fermentor studies lasting 70 h, the above engineered Halomonas TD01 grew to 112 g/L CDW containing 70 wt% P3HB, and to 80 g/L CDW with 70 wt% P(3HB-co-8 mol% 3HV) in the presence of propionic acid. The cells grown in shake flasks even accumulated close to 92% PHB in CDW with a significant increase of glucose to PHB conversion efficiency from around 30% to 42% after 48 h cultivation when pyridine nucleotide transhydrogenase was overexpressed. Halomonas TD01 was also engineered for producing a PHA regulatory protein PhaR which is a robust biosurfactant.     

Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway

Mesaconate is an intermediate in the glutamate degradation pathway of microorganisms such as Clostridium tetanomorphum. However, metabolic engineering to produce mesaconate has not been reported previously. In this work, two enzymes involved in mesaconate production, glutamate mutase and 3-methylaspartate ammonia lyase from C. tetanomorphum, were recombinantly expressed in Escherichia coli. To improve mesaconate production, reactivatase of glutamate mutase was discovered and adenosylcobalamin availability was increased. In addition, glutamate mutase was engineered to improve the in vivo activity. These efforts led to efficient mesaconate production at a titer of 7.81 g/L in shake flask with glutamate feeding. Then a full biosynthetic pathway was constructed to produce mesaconate at a titer of 6.96 g/L directly from glucose. In summary, we have engineered an efficient system in E. coli for the biosynthesis of mesaconate.     

Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species

Chitin, which is a polymer of β-(1–4) linked N-acetyl-d-glucosamine (GlcNAc) residues, is one of the most abundant renewable resources in nature, after cellulose. In this study, we found some native Mucor strains, which can use GlcNAc and chitin substrates as carbon sources for growth and ethanol production. One of these strains, M. circinelloides NBRC 6746 produced 18.6 ± 0.6 g/l of ethanol from 50 g/l of GlcNAc after 72 h and the maximum ethanol production rate was 0.75 ± 0.1 g/l/h. Furthermore, M. circinelloides NBRC 4572 produced 6.00 ± 0.22 and 0.46 ± 0.04 g/l of ethanol from 50 g/l of colloidal chitin and chitin powder after 16 and 12 days, respectively. We also found an extracellular chitinolytic enzyme producing strain M. ambiguus NBRC 8092, and successfully improved ethanol productivity of NBRC 4572 from colloidal chitin using crude chitinolytic enzyme derived from NBRC 8092. The ethanol titer reached 9.44 ± 0.10 g/l after 16 days. These results were the first bioethanol production from GlcNAc and chitin substrates by native organisms, and also suggest that these Mucor strains have great potential for the simultaneous saccharification and fermentation (SSF) of chitin biomass.     

High-level De novo biosynthesis of arbutin in engineered Escherichia coli

Arbutin is a hydroquinone glucoside compound existing in various plants. It is widely used in pharmaceutical and cosmetic industries owing to its well-known skin-lightening property as well as anti-oxidant, anti-microbial, and anti-inflammatory activities. Currently, arbutin is usually produced by plant extraction or enzymatic processes, which suffer from low product yield and expensive processing cost. In this work, we established an artificial pathway in Escherichia coli for high-level production of arbutin from simple carbon sources. First, a 4-hydroxybenzoate 1-hydroxylase from Candida parapsilosis CBS604 and a glucosyltransferase from Rauvolfia serpentina were characterized by in vitro enzyme assays. Introduction of these two genes into E. coli led to the production of 54.71 mg/L of arbutin from glucose. Further redirection of carbon flux into arbutin biosynthesis pathway by enhancing shikimate pathway genes enabled production of 3.29 g/L arbutin, which is a 60-fold increase compared with the initial strain. Final optimization of glucose concentration added in the culture medium was able to further improve the titer of arbutin to 4.19 g/L in shake flasks experiments, which is around 77-fold higher than that of initial strain. This work established de novo biosynthesis of arbutin from simple carbon sources and provided a generalizable strategy for the biosynthesis of shikimate pathway derived chemicals. The high titer achieved in our engineered strain also indicates the potential for industrial scale bio-manufacturing of arbutin.     

Investigations on alkaline protease production with B. subtilis PE-11 immobilized in calcium alginate gel beads

Bacillus subtilis PE-11 cells were immobilized in calcium alginate and used for the production of alkaline protease. The influence of alginate concentration, different cations, concentration of cation, curing time, bead diameter and nutrient strength on alkaline protease production and stability of biocatalyst were investigated. Repeated batch fermentations of immobilized cells in shake flasks were carried out with the optimized parameters such as; 3% alginate, 0.25 M calcium chloride with 1 h curing time, 3.24 mm bead diameter and 0.75% glucose and 0.75% peptone as nutrients. The results indicated that, a good level of enzyme was maintained for a period of about 9 days. The immobilized cells of B. subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation.     

Production of the postharvest biocontrol agent Bacillus subtilis CPA-8 using low cost commercial products and by-products

The aim of this research was to identify a low cost medium based on commercial products and by-products that provided maximum Bacillus subtilis CPA-8 growth and maintained biocontrol efficacy. Low cost media combining economical nitrogen and carbon sources such as yeast extract, peptone, soy products, sucrose, maltose and molasses were tested. Tests were carried out in 250-ml flasks containing 50 ml of each tested medium. Maximum cell growth (>3 × 10⁹ CFU ml^?1) was obtained in defatted soy flour 44% combined with sucrose or molasses media. Second, CPA-8 production was scaled up in a 5-l fermenter and CPA-8 population dynamics, pH and oxygen consumption in the optimized medium (defatted soy flour 44% – molasses) was recorded. In these tests, there was a 5-h lag phase before growth, after which exponential growth occurred and maximum production was 3 × 10⁹ CFU ml^?1 after 20 h. Fruit trials with cells and cell free supernatants from CPA-8 grown in optimized medium maintained biocontrol efficacy against Monilinia fructicola on peaches, resulting in disease reductions up to 95%. CPA-8 populations survived in wounds on inoculated peaches, regardless of the culture media used. The results show that B. subtilis CPA-8 can be produced in a low cost medium combining inexpensive nitrogen and carbon sources (40 g l^?1 defatted soy flour 44%, 5 g l^?1 molasses plus mineral trace supplements) in shake flasks and a laboratory fermenter (5 l). The results could be used to provide a reliable basis for scaling up the fermentation process to an industrial level.     

Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Enhanced solvent production by metabolic engineering of a twin-clostridial consortium

The efficient fermentative production of solvents (acetone, n-butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase (Clocel_1892) and lactate dehydrogenase (Clocel_1533), and to overexpress the gene encoding butyrate kinase (Clocel_3674), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene (Clocel_2243) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation (ctfAB, cbei_3833/3834) and pentose utilization (xylR, cbei_2385 and xylT, cbei_0109) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2 g/L of AECC and produce 22.1 g/L of solvents (4.25 g/L acetone, 11.5 g/L butanol and 6.37 g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.     

Evolutionary adaptation of Kluyveromyces marxianus strain for efficient conversion of whey lactose to bioethanol

The aim of the present work is to develop an osmotolerant yeast strain with high lactose utilization and further use it to ferment lactose rich whey permeate for high ethanol titer and to reduce energy consumption. Ethanol production and growth rate of selected MTCC 1389 strain were quite high in whey containing lactose up to 150 g/L but it remains constant in lactose concentration (200 g/L) as cells encountered osmotic stress. Thus, strain MTCC 1389 was used for an adaptation to lactose concentration 200 g/L for 65 days and used further for fermentation of lactose rich whey. Fermentation with an adapted K. marxianus MTCC 1389 strain in laboratory fermenter resulted in ethanol titer of 79.33 g/L which is nearly 17.5% higher than the parental strain (66.75 g/L). Expression analysis of GPD1, TPS1and TPS2 found upregulated in lactose adapted K. marxianus strain as compared to the parental strain. These results suggest that an adapted K. marxianus strain accumulates glycerol and trehalose in response to lactose stress and improve osmotolerance in K. marxianus cells. Thus, the study illustrates that evolutionary engineering is an efficient strategy to obtain a superior biofuel yeast strain, which efficiently ferments four-fold concentrated cheese whey.     

Modular optimization of multi-gene pathways for fumarate production

Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46 g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13 g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate.     

Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals

5-Aminovalerate (5AVA) is the precursor of valerolactam, a potential building block for producing nylon 5, and is a C5 platform chemical for synthesizing 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putida davAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured in a medium containing 20 g/L of glucose and 10 g/L of l-lysine, 3.6 g/L of 5AVA was produced by converting 7 g/L of l-lysine. When the davAB genes were introduced into recombinant E. coli strainXQ56allowing enhanced l-lysine synthesis, 0.27 and 0.5 g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate could be demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 20 g/L glucose, 10 g/L l-lysine and 10 g/L α-ketoglutarate, 1.7 g/L of glutarate was produced.     

Production of butyric acid by a cellulolytic actinobacterium Thermobifida fusca on cellulose

Thermobifida fusca not only produces cellulases, hemicellulases and xylanases, but also excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.     

Combining metabolic engineering and biocompatible chemistry for high-yield production of homo-diacetyl and homo-(S,S)-2,3-butanediol

Biocompatible chemistry is gaining increasing attention because of its potential within biotechnology for expanding the repertoire of biological transformations carried out by enzymes. Here we demonstrate how biocompatible chemistry can be used for synthesizing valuable compounds as well as for linking metabolic pathways to achieve redox balance and rescued growth. By comprehensive rerouting of metabolism, activation of respiration, and finally metal ion catalysis, we successfully managed to convert the homolactic bacterium Lactococcus lactis into a homo-diacetyl producer with high titer (95 mM or 8.2 g/L) and high yield (87% of the theoretical maximum). Subsequently, the pathway was extended to (S,S)-2,3-butanediol (S-BDO) through efficiently linking two metabolic pathways via chemical catalysis. This resulted in efficient homo-S-BDO production with a titer of 74 mM (6.7 g/L) S-BDO and a yield of 82%. The diacetyl and S-BDO production rates and yields obtained are the highest ever reported, demonstrating the promising combination of metabolic engineering and biocompatible chemistry as well as the great potential of L. lactis as a new production platform.     

The role of volumetric power input in the growth,morphology, and production of a recombinant glycoprotein by Streptomyces lividans in shake flasks

The impact of flask geometry on Streptomyces lividans growth and morphology, production and O-mannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries. During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51 kW/m³, followed by coiled flasks (CF) with 0.44 kW/m³ and normal Erlenmeyer flasks (NF) with 0.20 kW/m³ (flasks volume of 250 mL, filling with 50 mL and agitated at 150 rpm). During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology. Also, NF cultures were carried out at a filling volume and agitation of 15 mL, 150 rpm (15 mL-NF), and 25 mL, 168 rpm (25 mL-NF), in order to raise P/V closely to the values obtained in CF. However, different growth, morphology and recombinant protein productivity were obtained. These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production. But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V.