CRISPRi engineering E. coli for morphology diversification

Microbial morphology engineering has recently become interesting for biotechnology. Genes ftsZ and mreB encoding proteins of bacterial fission ring and skeletons, respectively, are essential for cell growth, they both are the most important genes keeping the bacterial shapes including the cell length and width, respectively. Clustered regularly interspaced short palindromic repeats interference, abbreviated as CRISPRi, was for the first time used in this study to regulate expression intensities of ftsZ or/and mreB in E. coli. Five sgRNAs associated with CRISPRi were designed and synthesized, respectively, to target five various locations on genes ftsZ or mreB encoded in the E. coli chromosome, resulting in various reduced expression levels of ftsZ or/and mreB, respectively, forming elongated or/and fatter cells. Repressions on gene expressions of ftsZ or/and mreB could be further intensified by combining various sgRNAs together. It was found that the stronger the repression on genes ftsZ or/and mreB, the longer the E. coli fibers, and the larger the E. coli cells. Combined repressions on expressions of ftsZ and mreB generated long and larger E. coli with diverse morphologies including various sizes of gourds, bars, coccus, spindles, multi-angles and ellipsoids. In all cases, accumulations of intracellular biopolyester polyhydroxybutyrate (PHB) were in direct proportional to the intracellular volumes, ranging from 40% to 80% PHB in bacterial cell dry weights, depending on the cell volumes increases by the above CRISPRi applications.     

Poly-β-hydroxybutyrate/ectoine co-production by ectoine-excreting strain Halomonas salina

The ectoine-excreting bacterial strain of Halomonas salina was employed in the co-production of poly-β-hydroxybutyrate (PHB) and ectoine (Ect) during a fermentation process (PHB/Ect co-production). An efficient PHB/Ect co-production process was carried out at low NaCl concentration (30 g L⁻¹). It was established using ¹H Nuclear Magnetic Resonance spectroscopy that H. salina produces PHB. The effects of the NaCl concentration, the initial C/N ratio, the phosphate concentration and mixed carbon sources were investigated with respect to PHB/Ect co-production. The PHB/Ect co-production system comprised growing and non-growing cell phases and was developed with NaCl concentration of 30 g L⁻¹. The optimal conditions for PHB/Ect co-production by the ectoine-excreting strain of H. salina were 30 g L⁻¹ NaCl, with an initial C/N ratio of 15, an initial phosphate concentration of 12 g L⁻¹ and mixed carbon sources of 55 g L⁻¹ glucose and 25 g L⁻¹ monosodium glutamate. Using a PHB/Ect co-production system with growing and non-growing cell phases prevents the inhibition of PHB synthesis by high concentration of NaCl and significantly reduces ectoine degradation. PHB and ectoine concentrations as high as 35.3 g L⁻¹ and 8.6 g L⁻¹, respectively, were achieved. The efficient co-production of PHB and ectoine at a low NaCl concentration has been realised.     

Statistical media optimization for growth and PHB production from methanol by a methylotrophic bacterium

Media components were optimized by statistical design for cell growth and PHB production of Methylobacterium extorquens DSMZ 1340. Four important components of growth media were optimized by central composite design. The growth increased from an OD = 1.35 for Choi medium as control to an OD = 2.15 for optimal medium. Then media components for PHB production were optimized. Optimization of five important factors was conducted by response surface method. The optimal composition of PHB production medium was found to be at 7.8 (g/L) Na₂HPO₄ · 12H₂O, and surprisingly at zero concentration of (NH₄)₂SO₄, KH₂PO₄, MgSO₄ and MnSO₄. The PHB production was found to be 2.95 (g/L) at this medium. RSM results indicated that a deficiency of nitrogen and magnesium is crucial for PHB accumulation in this microorganism. Also, PHB production was carried out in a 5 L fermentor at the optimum condition which resulted in 9.5 g/L PHB and 15.4 g/L cell dry weight with 62.3% polymer content.     

Vibrio taketomensis sp. nov. by genome taxonomy

Two novel strains C4III282^T and C4III291 were isolated from seawater collected a site off the Taketomi coral reef. Phylogenetic analysis based on the 16S rRNA sequences revealed that the two strains belong to the genus Vibrio. MLSA using eight protein-coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) showed that C4III282^T and C4III291 are closely related to the members of the Ponticus clade, namely Vibrio panuliri JCM 19500^T, Vibrio ponticus DSM 16217^T, and “Vibrio rhodolitus” G98. ANI and in silico DDH values with members of the Ponticus clade were 77.6-78.7% and 22.2-23.1, respectively. The name Vibrio taketomensis sp. nov. is proposed with C4III282^T (CAIM 1928^T = DSM 106943^T = JCM 33434^T) as the type strain.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus

This work investigated effects of lignocellulose degradation products on cell biomass and lipid production by Cryptococcus curvatus. Furfural was found to have the strongest inhibitory effect. For the three phenolic compounds tested, vanillin was the most toxic, while PHB and syringaldehyde showed comparable inhibitions in the concentration range of 0–1.0 g/L. Generally little significant differences on the relative cell biomass and lipid contents at the same concentrations of tested compounds were observed between glucose and xylose as a sole carbon source. At 1.0 g/L of furfural, the cell biomass and lipid content decreased by 78.4% and 61.0% for glucose as well as 72.0% and 59.3% for xylose, respectively. C. curvatus ceased to grow at concentrations of PHB over 1.0 g/L or vanillin over 1.5 g/L. The strain could survive in the presence of syringaldehyde up to 2.0 g/L for glucose or 1.5 g/L for xylose. The compounds’ negative impact was reduced by an increase in inoculum size and a 10% (v/v) seed was detected to be optimal for cell biomass and lipid production. The results demonstrated C. curvatus could effectively utilize most of the dominant monosaccharides and cellobiose existing in lignocellulosic biomass hydrolysate in the presence of toxic compounds.     

Efficiency of energy use for pregnancy by meat goat does with different litter size

Twenty-four Boer × Spanish does (3 years of age, having kidded once previously and with an initial BW of 42.7 ± 1.2 kg) were used to determine the efficiency of ME utilization for pregnancy (k_preg). Six does were nonpregnant and, based on ultrasound determination on day 45 of gestation, six had a litter size (LS) of 1, 2, and 3. However, only 10 of the pregnant does delivered the expected number of kids (3, 4, and 3 with LS of 1, 2, and 3, respectively). Does were fed a diet of approximately 50% concentrate in accordance with assumed maintenance plus pregnancy energy requirements based on estimated nonpregnancy tissue BW and LS. Recovered energy (RE) was determined by subtraction of energy expenditure (EE; respiration calorimetry) near days 80, 100, 120, and 140 of gestation from ME intake (MEI). RE was assumed attributable to pregnancy tissues (fetus, fetal fluids and membranes, uterus, and mammary gland), and ME used for pregnancy (ME_preg) was estimated by subtracting ME_m determined with nonpregnant goats from MEI by those pregnant. For does with actual LS equal to that expected, the no-intercept equation for the regression of RE against ME_preg was: RE = ME_preg × 0.252 (S.E. = 0.030; R² = 0.64), indicating a k_preg of 25%. A regression including LS (1 versus 2 or 3) suggested greater k_preg for LS of 1 (40.2 ± 5.6%) versus 2 or 3 (20.5 ± 3.2%). Regressions for goats with LS different from expected suggested positive effects of use of energy mobilized from nonpregnancy tissues on k_preg and of use of dietary ME for energy accretion in nonpregnancy tissues on the efficiency of whole body ME utilization. In conclusion, the average efficiency of ME use for pregnancy regardless of LS in goats was near 25%, which when considering the expected proportion of all pregnancy tissues attributable to fetal or conceptus tissues implies an energy requirement for pregnancy of goats similar to common recommendations for sheep and cattle.     

Microbial production of poly-β-hydroxybutyrate by marine microbes isolated from various marine environments

Considering the industrial interest of Poly-β-hydroxybutyrate (PHB), bacteria isolated from the various marine arenas were screened for their ability to accumulate PHB and were compared with Wausteria eutropha (MTCC-1285). Among the 42 isolates, four strains showed the accumulation of PHB. The maximum PHB producer Vibrio sp. (MK4) was further studied in detail. To increase the productivity, steps were taken to evaluate the effect of carbon sources, nitrogen sources, pH and sodium chloride concentration on PHB productivity by MK4. The optimized conditions were further used for the batch fermentation over a period of 72 h. Significantly higher maximum biomass of 9.1 g/L with a PHB content of 4.223 g/L was obtained in a laboratory-scale bioreactor at 64 h, thus giving a productivity of 0.065 g/L/h. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis and ¹H NMR analysis. Thus, the study highlights the potential of the use of Vibrio sp (MK4) in the commercial production of PHB.     

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO₂: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670 mg/L total hydrocarbons containing 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO₂ as the sole carbon source.     

Thermophilic bacterium Caldimonas taiwanensis produces poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from starch and valerate as carbon sources

Caldimonas taiwanensis accumulated polyhydroxybutyrate (PHB) at 55 °C from gluconate, fructose, maltose, and glycerol under nitrogen-limited condition. The PHB content peaked at 14 h after inoculation from gluconate. C. taiwanensis did not grow or accumulate PHA from fatty acids as the sole carbon source; however, it incorporated 3-hydroxyvalerate (3-HV) into PHB polymer from gluconate and valerate as a mixed carbon source. By adjusting the valerate concentration, the molar fraction of 3-HV could be modulated from 10 mol% to 95 mol%. Fatty acid valerate substantially inhibited cell growth and PHA accumulation with the addition of as little as 5 mM to the medium. Supplementing the medium with yeast extract overcame the inhibition, which enhanced not only the yield of biomass but also PHA productivity. The in vivo substrate specificity of PHA synthase ranged from C₄ to C₆. In addition, C. taiwanensis also incorporated a wide range of 3-HV into PHA from soluble starch and valerate as a mixed carbon source. Food-grade starches made from cassava, corn, potato, sweet potato and wheat respectively mixed with valerate were studied for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production. In this study, C. taiwanensis exhibited high promise for reducing the production cost of P(3HB-co-3HV).     

Poly(β-hydroxybutyrate) production by a moderate halophile,Halomonas boliviensis LC1

The moderate halophile Halomonas boliviensis, isolated from a Bolivian saline soil sample, was able to accumulate poly(β-hydroxybutyrate) (PHB) when grown under conditions of nutrient limitation and excess carbon source. The concentration of sodium chloride in the medium influenced the cell-growth, -size, and rate of PHB accumulation. Cultivation in shake flasks led to a PHB accumulation of about 54 wt.% with respect to cell dry weight at 4.5% (w/v) NaCl in a medium with butyric acid and sodium acetate as carbon sources. The production of PHB was substantially improved to a maximum value of 88 wt.% during cultivation under controlled conditions of pH and oxygen concentration in a fermentor. The use of glucose and sucrose, respectively, as carbon source could also lead to the production of PHB at an average level of 55 wt.%.     

Microbial biodegradable plastic production from a wheat-based biorefining strategy

Restructuring the current fermentation and recovery practices employed for the production of polyhydroxyalkanoates is essential for the commercialisation of environmentally benign and cost competitive biodegradable plastics. This study presents the potential of a wheat-based biorefinery for the production of poly(3-hydroxybutyrate) (PHB). Fed-batch bioconversions using Wautersia eutropha growing on wheat-derived media led to the production of 162.8 g/l PHB. A high PHB to total dry weight (TDW) yield of 93% (w/w) was achieved due to microbial autolysis at the end of fermentation. Images of bacterial cells taken with a Transmission Electron Microscope (TEM) indicated the potential of bacterial autolysis as a mean to shorten downstream processing for PHB purification. The consumption of amino acids and peptides derived from wheat gluten hydrolysis resulted in a high glucose to PHB conversion yield of 0.47 g/g. The respective yield regarding the amount of wheat used for the production of enzymes and PHB was around 0.3 g PHB/g wheat, which corresponds to 82.8% of the maximum theoretical conversion yield. The productivity achieved was around 0.9 g/l h. Fermentations carried out on wheat-derived media and media formulated with various commercial sources of nutrients (glucose, yeast extract, soy-protein acid hydrolysate, casein hydrolysates, corn steep liquor and various inorganic chemicals) showed that the proposed wheat-based biorefinery strategy enhanced PHB production.     

Production and characterization of terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaAB

Recombinant Aeromonas hydrophila 4AK4 harboring phbA and phbB (phaAB) genes encoding β-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha produced a terpolyester of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from mixtures of dodecanoic acid and propionic acid. Depending on the concentration of propionic acid in bacterial cultures, cell growth represented by cellular dry weight (CDW), P(3HB-co-3HV-co-3HHx) contents in dry cells and 3HV molar percentage in the terpolyester ranged from 0.43 g l⁻¹ to 3.29 g l⁻¹, 20.7% to 35.6%, 2.3 mol% to 7.1 mol%, respectively. Number average molecular (M_n) weights of the terpolyesters were 303,000–800,000, independent from monomer fraction content. This terpolyester was characterized by nuclear magnetic resonance (NMR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurement studies. Results showed that the terpolyester had higher thermal stability and elongation at break compared with that of homopolymer poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB-co-5 mol%3HV) or P(3HB-co-12 mol%3HHx). In addition, the terpolyester had lower melting (T_m) temperatures and enthalpy of fusions (ΔH_m) than PHB did.     

Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris

The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265 U/L and 300 mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145 U/L and 200 mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100 U/L) and protein yield (2.0 g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.     

Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38 ± 0.64 × 10^?5. The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75 ± 2.7% and 5–6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45 mM of NaAsO₂. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.     

Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates

The halophile Halomonas TD01 and its derivatives have been successfully developed as a low-cost platform for the unsterile and continuous production of chemicals. Therefore, to increase the genetic engineering stability of this platform, the DNA restriction/methylation system of Halomonas TD01 was partially inhibited. In addition, a stable and conjugative plasmid pSEVA341 with a high-copy number was constructed to contain a LacI^q-P_trc system for the inducible expression of multiple pathway genes. The Halomonas TD01 platform, was further engineered with its 2-methylcitrate synthase and three PHA depolymerases deleted within the chromosome, resulting in the production of the Halomonas TD08 strain. The overexpression of the threonine synthesis pathway and threonine dehydrogenase made the recombinant Halomonas TD08 able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV consisting of 4–6 mol% 3-hydroxyvalerate or 3HV, from various carbohydrates as the sole carbon source. The overexpression of the cell division inhibitor MinCD during the cell growth stationary phase in Halomonas TD08 elongated its shape to become at least 1.4-fold longer than its original size, resulting in enhanced PHB accumulation from 69 wt% to 82 wt% in the elongated cells, further promoting gravity-induced cell precipitations that simplify the downstream processing of the biomass. The resulted Halomonas strains contributed to further reducing the PHA production cost.     

Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli

A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588) — a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240 AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.     

Analysis of the germination rate,mitotic index and karyotype of Chromolaena barranquillensis (Hieron.) R.M. King & H. Rob.—Asteraceae

Chromolaena barranquillensis (Asteraceae) is an endemic plant of northern Colombia that has garnered economic and medicinal interest, because species from the genus Chromolaena have shown diverse biological activities. This study describes, for the first time, the karyotype, germination and mitotic indices of C. barranquillensis (Hieron.) R.M. King & H. Rob. The germination index was between 34% and 56% with an average germination rate of 1.2 ± 0.4 seeds/day. The mitotic index analysis allowed to determine the cell cycle time (4 h, 10 min) and the mitotic hours (3:00–8:00 h and 17:00 h). The mitosis time was 49 min, equivalent to ~ 20% of the cell cycle. Karyotype analysis showed that C. barranquillensis is a hexaploid species with a chromosomal formula 2n = 6x = 60 = 48 m + 12 sm, and the average chromosomal lengths were 1.7 ± 0.1 μm to 0.9 ± 0.3 μm. The Stebbins asymmetry index was 2B, and the total form percentage was ~ 41%. These results uncover differences between C. barranquillensis and Chromolaena odorata, one of the most abundant species found in the world and the most closely related species to C. barranquillensis.