<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of oleaginous yeast <Emphasis Type="Italic">Lipomyces</Emphasis> species

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.     

Optimization of C16 and C18 fatty alcohol production by an engineered strain of <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The oleaginous yeast Lipomyces starkeyi was engineered for the production of long-chain fatty alcohols by expressing a fatty acyl-CoA reductase, mFAR1, from Mus musculus. The optimal conditions for production of fatty alcohols by this strain were investigated. Increased carbon-to-nitrogen ratios led to efficient C16 and C18 fatty alcohol production from glucose, xylose and glycerol. Batch cultivation resulted in a titer of 1.7 g/L fatty alcohol from glucose which represents a yield of 28 mg of fatty alcohols per gram of glucose. This relatively high level of production with minimal genetic modification indicates that L. starkeyi may be an excellent host for the bioconversion of carbon-rich waste streams, particularly lignocellulosic waste, to C16 and C18 fatty alcohols.     

Reprogramming <Emphasis Type="Italic">Halomonas</Emphasis> for industrial production of chemicals

Halomonas spp. are able to grow under a high salt concentration at alkali pH, they are able to resist contamination by other microbes. Development of Halomonas spp. as platform production strains for the next-generation industrial biotechnology (NGIB) is intensively studied. Among Halomonas spp., Halomonas bluephagenesis is the best studied one with available engineering tools and methods to reprogram it for production of various polyhydroxyalkanoates, proteins, and chemicals. Due to its contamination resistance, H. bluephagenesis can be grown under open and continuous processes not just in the labs but also in at least 1000 L fermentor scale. It is expected that NGIB based on Halomonas spp. be able to engineer for production of increasing number of products in a competitive manner.     

Expression of an endoglucanase–cellobiohydrolase fusion protein in <Emphasis Type="Italic">Saccharomyces cerevisiae,Yarrowia lipolytica,</Emphasis> and <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The low secretion levels of cellobiohydrolase I (CBHI) in yeasts are one of the key barriers preventing yeast from directly degrading and utilizing lignocellulose. To overcome this obstacle, we have explored the approach of genetically linking an easily secreted protein to CBHI, with CBHI being the last to be folded. The Trichoderma reesei eg2 (TrEGII) gene was selected as the leading gene due to its previously demonstrated outstanding secretion in yeast. To comprehensively characterize the effects of this fusion protein, we tested this hypothesis in three industrially relevant yeasts: Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. Our initial assays with the L. starkeyi secretome expressing differing TrEGII domains fused to a chimeric Talaromyces emersonii–T. reesei CBHI (TeTrCBHI) showed that the complete TrEGII enzyme, including the glycoside hydrolase (GH) 5 domain is required for increased expression level of the fusion protein when linked to CBHI. We found that this new construct (TrEGII–TeTrCBHI, Fusion 3) had an increased secretion level of at least threefold in L. starkeyi compared to the expression level of the chimeric TeTrCBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in S. cerevisiae and Y. lipolytica. Digestion of pretreated corn stover with the secretomes of Y. lipolytica and L. starkeyi showed that conversion was much better using Y. lipolytica secretomes (50% versus 29%, respectively). In Y. lipolytica, TeTrCBHI performed better than the fusion construct. Furthermore, S. cerevisiae expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, pNP-cellobiose. No activity was observed for the pNP-lactose substrate. Clearly, this approach is not universally applicable to all yeasts, but works in specific cases. With purified protein and soluble substrates, the exoglucanase activity of the GH7 domain embedded in the Fusion 3 construct in L. starkeyi was significantly higher than that of the GH7 domain in TeTrCBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just TeTrCBHI. We conclude that the strategy of leading TeTrCBHI expression with a linked TrEGII module significantly improved the expression of active CBHI in L. starkeyi.     

Growth and wax ester production of an <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis> ADP1 mutant deficient in exopolysaccharide capsule synthesis

Acinetobacter baylyi ADP1 naturally produces wax esters that could be used as a raw material in industrial applications. We attempted to improve wax ester yield of A. baylyi ADP1 by removing rmlA, a gene involved in exopolysaccharide production. Growth rate, biomass formation and wax ester yield on 4-hydroxybenzoate were not affected, but the rmlA ^? strain grew slower on acetate, while reaching similar biomass and wax ester yield. The rmlA ^? cells had malformed shape and large size and grew poorly on glucose without expression of the gene for pyruvate kinase (pykF) from Escherichia coli. The pykF-expressing rmlA ^? strain had similar growth rate, lowered biomass formation and improved wax ester production on glucose as compared to the wild-type strain expressing pykF. Cultivation of the pykF-expressing rmlA ^? strain on an elevated glucose concentration in a medium supplemented with amino acids resulted in doubled molar wax ester yield and acetate production.     

Production of optically pure 2,3-butanediol from <Emphasis Type="Italic">Miscanthus floridulus</Emphasis> hydrolysate using engineered <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strains

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L d-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L d-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L d-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.     

Camptothecin from <Emphasis Type="Italic">Nothapodytes nimmoniana</Emphasis>: review on biotechnology applications

Since the first report on Camptothecin detection in Nothapodytes nimmoniana by Govindachari and Viswanathan (Phytochem 11:35–29, 1972), considerable work has been done on biotechnology and its applications on the species. Plant tissue culture techniques have applications in clonal propagation, CPT production, and conservation of N. nimmoniana. Discovery of CPT production by endophytes existing in symbiotic association with N. nimmoniana has provided new insights into finding alternative sources of the alkaloid. Development of molecular markers such as RFLP, RAPD, ISSR, and AFLP has facilitated understanding of population ecology and genetics of the species. Molecular information generated from these studies is promising in establishing strategies for conservation and sustainable use of N. nimmoniana populations under overexploitation pressure. The advances in instrumentation in the 20th century, such as desorption electrospray ionization mass spectrometry allowed CPT analysis in tissues without sample pretreatment. Other ancient techniques for qualitative and quantitative analysis such as chromatography, spectroscopy, and H1-NMR are applied in the detection of CPT due to variable sensitivity to the alkaloid. The review covers work on plant tissue culture for clonal propagation and CPT production in N. nimmoniana. Besides symbiotic endophyte sources of CPT in N. nimmoniana, population genetics studies and instrumentation analysis of the alkaloids are reviewed.     

Cytoplasmic expression of a thermostable invertase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.

Results

The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.

Conclusions

Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

     

Enhancement of Pyruvate Productivity in <Emphasis Type="Italic">Candida glabrata</Emphasis> by Deleting the <Emphasis Type="Italic">CgADE13</Emphasis> Gene to Improve Acid Tolerance

Acid tolerance is one of the critical factors to evaluate the quality of the industrial production strains, especially organic acid producing microorganisms. To circumvent this problem, we investigated the physiological function of adenylosuccinate lyase in AMP metabolism from Candida glabrata by deleting the corresponding gene, CgADE13. At pH 4.0, CgADE13 deletion resulted in a 68.3% and 112.0% increase in biomass and cell viability compared to those of wild type strain (wt), respectively. In addition, CgADE13 deletion also protected cell morphology and counteracted ROS production. Further, the intracellular ATP level of strain Cgade13Δ was decreased by 25.0%, and its H+-ATPase activity was increased by 15.0%. Finally, pyruvate production with strain Cgade13Δ in a 30-L batch bioreactor at pH 4.0 reached 53.9 g/L, and pyruvate productivity was increased by 166.7% compared to that of wt. This is the first report regarding tolerance engineering of C. glabrata for enhancing pyruvate productivity, which provides a good starting point for metabolic engineering to achieve the industrial production of other chemicals.     

Identification of alcohol stress tolerance genes of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 using adaptive laboratory evolution

Background

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.

Results

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Conclusions

Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.

     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

<Emphasis Type="Italic">glyA</Emphasis> gene knock-out in <Emphasis Type="Italic">Escherichia coli</Emphasis> enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C₁ units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C₁ supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA ^Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.     

New recombinant strains of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> with overexpression of the aconitate hydratase gene for the obtainment of isocitric acid from rapeseed oil

The yeast Yarrowia lipolytica is capable of high-intensity synthesis (overproduction) of citric (CA) and isocitric (ICA) acids under nitrogen limitation. The ratio of the synthesized acids depends on the producing strains used and the expression level of the aconitate hydratase gene (ACO1). Recombinant variants with overexpression of the multicopy ACO1 gene have been obtained based on the natural ICA-producing strain Y. lipolytica 672. A recombinant strain Y. lipolytica 20, which has an isocitrate-citrate ratio shifted towards ICA (2.3: 1) as compared to the parental strain (1.1: 1), has been selected. Culturing of the 20 variant in a 10 L reactor has resulted in the production of 72.6 g/L of ICA and 29.0 g/L of CA with a ratio of 2.5: 1. This makes it possible to regard Y. lipolytica 20 as a promising producer for the development of an industrial process for isocitrate production.     

Analysis of antimicrobial and immunomodulatory substances produced by heterofermentative <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Antimicrobial and immunomodulatory potential of various Lactobacillus reuteri strains is closely connected to their metabolite production profile under given cultivation conditions. We determined the in vitro production of antimicrobial substances such as organic acids, ethanol, and reuterin by four strains of L. reuteri (L. reuteri E, L. reuteri KO5, L. reuteri CCM 3625, and L. reuteri ATCC 55730). All studied L. reuteri strains showed the ability to produce lactic acid, acetic acid, and ethanol with concominant consumption of glucose and together with phenyllactic acid—a potent antifungal compound—with concominant consumption of phenylalanine. The reuterin production from glycerol was confirmed for all analyzed lactobacilli strains except L. reuteri CCM 3625. Production of organic acids, ethanol, and reuterin is significantly involved in antimicrobial activity of lactobacilli which was determined using the dual-culture overlay diffusion method against six indicator bacteria and five indicator moulds. In comparison to the referential L. reuteri ATCC 55730, the highest inhibition potential was observed against Escherichia coli CCM 3988 and Pseudomonas aeruginosa CCM 3955. Among analyzed indicators of moulds, the growth of Alternaria alternata CCM F-128 was the most inhibited by all four analyzed L. reuteri strains. Finally, the immunomodulatory potential of analyzed lactobacilli were proven by the determination of the in vitro production of biogenic amines histamine and tyramine. L. reuteri CCM 3625 was able to produce tyramine, and L. reuteri E and L. reuteri KO5 were able to produce histamine under given cultivation conditions.     

Expression and Characterization of Glucose Oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.     

Metabolic engineering of lipid pathways in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and staged bioprocess for enhanced lipid production and cellular physiology

Microbially produced lipids have attracted attention for their environmental benefits and commercial value. We have combined lipid pathway engineering in Saccharomyces cerevisiae yeast with bioprocess design to improve productivity and explore barriers to enhanced lipid production. Initially, individual gene expression was tested for impact on yeast growth and lipid production. Then, two base strains were prepared for enhanced lipid accumulation and stabilization steps by combining DGAT1, ΔTgl3 with or without Atclo1, which increased lipid content ~?1.8-fold but reduced cell viability. Next, fatty acid (FA) biosynthesis genes Ald6-SEACS^L641P alone or with ACC1** were co-expressed in base strains, which significantly improved lipid content (8.0% DCW, 2.6-fold than control), but severely reduced yeast growth and cell viability. Finally, a designed two-stage process convincingly ameliorated the negative effects, resulting in normal cell growth, very high lipid productivity (307 mg/L, 4.6-fold above control) and improved cell viability.     

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor—staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the β-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the β-phaseolin promoter.