An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Background

Biodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.

Results

A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings.

Conclusions

The results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

     

Metabolic engineering for high glycerol production by the anaerobic cultures of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.     

Dynamics and diversity of a microbial community during the fermentation of industrialized <Emphasis Type="Italic">Qingcai</Emphasis> paocai,a traditional Chinese fermented vegetable food,as assessed by Illumina MiSeq sequencing,DGGE and qPCR assay

Paocai is a traditional Chinese fermented food and typically produced via spontaneous fermentation. We have investigated the microbial community utilized for the fermentation of industrialized Qingcai paocai using the combination of Illumina MiSeq sequencing, PCR-mediated denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) assay. Three main phyla, namely Firmicutes, Proteobacteria and Bacteroidetes, were identified by both MiSeq sequencing and PCR-DGGE. The dominant genera observed in the fermentation were Lactobacillus, Pseudomonas, Vibrio and Halomonas. Most genera affiliated with Proteobacteria or Bacteroidetes were detected more often during the earlier part of the fermentation, while Lactobacillus (affiliated with Firmicutes) was dominant during the later fermentation stages. Fungal community analysis revealed that Debaryomyces, Pichia and Kazachstania were the main fungal genera present in industrialized Qingcai paocai, with Debaryomyces being the most dominant during the fermentation process. The quantities of dominant genera Lactobacillus and Debaryomyces were monitored using qPCR and shown to be 10⁹–10¹² and 10⁶–10¹⁰ copies/mL, respectively. During the later fermentation process of industrialized Qingcai paocai, Lactobacillus and Debaryomyces were present at 10¹¹ and 10⁸ copies/mL, respectively. These results facilitate further understanding of the unique microbial ecosystem during the fermentation of industrialized Qingcai paocai and guide future improvement of the fermentation process.     

Diversity and dynamics of lactic acid bacteria in <Emphasis Type="Italic">Atole agrio</Emphasis>, a traditional maize-based fermented beverage from South-Eastern Mexico,analysed by high throughput sequencing and culturing

The purpose of this work was to analyse the diversity and dynamics of lactic acid bacteria (LAB) throughout the fermentation process in Atole agrio, a traditional maize based food of Mexican origin. Samples of different fermentation times were analysed using culture-dependent and -independent approaches. Identification of LAB isolates revealed the presence of members of the genera Pediococcus, Weissella, Lactobacillus, Leuconostoc and Lactococcus, and the predominance of Pediococcus pentosaceus and Weissella confusa in liquid and solid batches, respectively. High-throughput sequencing (HTS) of the 16S rRNA gene confirmed the predominance of Lactobacillaceae and Leuconostocaceae at the beginning of the process. In liquid fermentation Acetobacteraceae dominate after 4 h as pH decreased. In contrast, Leuconostocaceae dominated the solid fermentation except at 12 h that were overgrown by Acetobacteraceae. Regarding LAB genera, Lactobacillus dominated the liquid fermentation except at 12 h when Weissella, Lactococcus and Streptococcus were the most abundant. In solid fermentation Weissella predominated all through the process. HTS determined that Lactobacillus plantarum and W. confusa dominated in the liquid and solid batches, respectively. Two oligotypes have been identified for L. plantarum and W. confusa populations, differing in a single nucleotide position each. Only one of the oligotypes was detected among the isolates obtained from each species, the biological significance of which remains unclear.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and metabolic activators: HXT3 gene expression and fructose/glucose discrepancy in sluggish fermentation conditions

When exposed to mixtures of glucose and fructose, as occurs during the fermentation of grape juice into wine, Saccharomyces cerevisiae uses these sugars at different rates. Moreover, glucose and fructose are transported by the same hexose transporters (HXT), which present a greater affinity for glucose, so that late in fermentation, fructose becomes the predominant sugar. Only a few commercial fermentation activators are available to optimally solve the problems this entails. The aim of this study was to investigate the relation between HXT3 gene expression and fructose/glucose discrepancy in two different media inoculated with a commercial wine strain of S. cerevisiae in the presence of three metabolic activators. Fermentation kinetics, vitality and major metabolites were also measured. Rehydration with ergosterol improved the area under the curve and the growth rate (µ _max ) in both studied media. Also, the fructose/glucose discrepancy values were improved with all activator treatments, highlighting rehydration in the presence of ascorbic acid. The yeast rehydration process was demonstrated to influence HXT3 expression under the studied conditions. Tetrahydrofolic acid treatment greatly influenced HXT3 gene expression, especially on the 12th day of the fermentation process. To a lesser extent, ergosterol and ascorbic acid also improved this parameter.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

Constitutive expression of rice <Emphasis Type="Italic">ORGAN SIZE RELATED</Emphasis> genes in <Emphasis Type="Italic">Arabidopsis</Emphasis> results in organ size enlargement

In plants, organ size control is a fundamental process during development. The Arabidopsis ORGAN SIZE RELATED (OSR) gene family plays a key role in organ size regulation. To explore the roles of OSR orthologs in rice, a BLAST search in the rice genome was performed and five putative OSR orthologs were isolated and designated as OsOSR. Constitutive expression of OsOSR1, OsOSR2 and OsOSR4 in Arabidopsis resulted in enlarged organ sizes, as a consequence of enhanced cell number and cell size, while the increase of organ size in the OsOSR3 and OsOSR5-expressing plants was only due to cell enlargement. Our results suggest that the rice OsOSR genes possess the conserved organ growth-promoting function and may be involved in the coordination of cell proliferation and expansion during plant development.     

Production of <Emphasis Type="Italic">cis</Emphasis>-Vaccenic Acid-oriented Unsaturated Fatty Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesel is produced worldwide as an alternative energy fuel and substitute for petroleum. Biodiesel is often obtained from vegetable oil, but production of biodiesel from plants requires additional land for growing crops and can affect the global food supply. Consequently, it is necessary to develop appropriate microorganisms for the development of an alternative biodiesel feedstock. Escherichia coli is suitable for the production of biodiesel feedstocks since it can synthesize fatty acids for lipid production, grows well, and is amenable to genetic engineering. Recombinant E. coli was designed and constructed for the production of biodiesel with improved unsaturated fatty acid contents via regulation of the FAS pathway consisting of initiation, elongation, and termination steps. Here, we investigated the effects of fabA, fabB, and fabF gene expression on the production of unsaturated fatty acids and observed that the concentration of cis-vaccenic acid, a major component of unsaturated fatty acids, increased 1.77-fold compared to that of the control strain. We also introduced the genes which synthesize malonyl-ACP used during initiation step of fatty acid synthesis and the genes which produce free fatty acids during termination step to study the effect of combination of genes in elongation step and other steps. The total fatty acid content of this strain increased by 35.7% compared to that of the control strain. The amounts of unsaturated fatty acids and cis-vaccenic acid increased by 3.27 and 3.37-fold, respectively.     

<Emphasis Type="Italic">In situ</Emphasis> fermentation dynamics during production of <Emphasis Type="Italic">gundruk</Emphasis> and <Emphasis Type="Italic">khalpi</Emphasis>, ethnic fermented vegetable products of the Himalayas

Gundruk is a fermented leafy vegetable and khalpi is a fermented cucumber product, prepared and consumed in the Himalayas. In situ fermentation dynamics during production of gundruk and khalpi was studied. Significant increase in population of lactic acid bacteria (LAB) was found during first few days of gundruk and khlapi fermentation, respectively. Gundruk fermentation was initiated by Lactobacillus brevis, Pediococcus pentosaceus and finally dominated by Lb. plantarum. Similarly in khalpi fermentation, heterofermentative LAB such as Leuconostoc fallax, Lb. brevis and P. pentosaceus initiated the fermentation and finally completed by Lb. plantarum. Attempts were made to produce gundruk and khalpi using mixed starter culture of LAB previously isolated from respective products. Both the products prepared under lab condition had scored higher sensory-rankings comparable to market products.     

Engineering <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> for the production of cyclopropanated fatty acids

Traditional synthesis of biodiesel competes with food sources and has limitations with storage, particularly due to limited oxidative stability. Microbial synthesis of lipids provides a platform to produce renewable fuel with improved properties from various renewable carbon sources. Specifically, biodiesel properties can be improved through the introduction of a cyclopropane ring in place of a double bond. In this study, we demonstrate the production of C19 cyclopropanated fatty acids in the oleaginous yeast Yarrowia lipolytica through the heterologous expression of the Escherichia coli cyclopropane fatty acid synthase. Ultimately, we establish a strain capable of 3.03?±?0.26 g/L C19 cyclopropanated fatty acid production in bioreactor fermentation where this functionalized lipid comprises over 32% of the total lipid pool. This study provides a demonstration of the flexibility of lipid metabolism in Y. lipolytica to produce specialized fatty acids.     

Characterization of<Emphasis Type="SmallCaps"> D</Emphasis>-lactic acid,spore-forming bacteria and <Emphasis Type="Italic">Terrilactibacillus laevilacticus</Emphasis> SK5-6 as potential industrial strains

In this study, we screened and isolated D-lactic acid-producing bacteria from soil and tree barks collected in Thailand. Among the isolates obtained, Terrilactibacillus laevilacticus SK5-6 exhibited good D-lactate production in the primary screening fermentation (99.27 g/L final lactate titer with 0.90 g/g yield, 1.38 g/L?h, and 99.00% D-enantiomer equivalent). Terrilactibacillus laevilacticus SK5-6 is a Gram-positive, endospore-forming, homofermentative D-lactate producer that can ferment a wide range of sugars to produce D-lactate. Unlike the typical D-lactate producers, such as catalase-negative Sporolactobacillus sp., T. laevilacticus SK5-6 possesses catalase activity; therefore, a two-phase fermentation was employed for D-lactate production. During an aerobic preculture stage, a high-density cell mass was rapidly obtained due to aerobic respiration. When transferred to the fermentation stage at the correct physiological stage (inoculum age) and proper concentration of cell mass (inoculum size), T. laevilacticus rapidly converted glucose into D-lactate under anaerobic conditions, resulting in a high final lactate titer (102.22 g/L), high yield (0.84 g/g), and high productivity (2.13 g/L?h). When the process conditions were shifted from an aerobic to an anaerobic environment, unlike other lactate-producing bacteria, the mixed acid fermentation route was not activated in the culture of T. laevilacticus SK5-6 during the fermentation stage when some trace oxygen still remained. Our study demonstrates the excellent characteristics of this isolate for D-lactate production; in particular, a high product yield was obtained without byproduct formation. Based on these key characteristics of T. laevilacticus SK5-6, we suggest that this isolate is a novel D-lactate producer for use in industrial fermentation.     

Regulation of NADH Oxidase Expression <Emphasis Type="Italic">via</Emphasis> a Thermo-regulated Genetic Switch for Pyruvate Production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

As a chemical, pyruvate can be used as a raw material for drug, agrochemical, chemical, and food industries. In the microbial production of pyruvate, although continuous expression of exogenous NADH oxidase (noxE) can improve glucose consumption, it can lead to a decrease of pyruvate yield. For efficient pyruvate production, a thermo-regulated genetic switch was designed to dynamically control the expression of noxE from Lactococcus lactis on the Escherichia coli MP-XB010CN chromosome. At the initial stage of fermentation, switching on the genetic switch for efficient noxE expression can promote growth rate and biomass accumulation, then switching off noxE expression can weaken the TCA pathway and improve the pyruvate yield. High pyruvate concentration of 93.0 g/L and yield of 0.71 g/g glucose were achieved with the thermo-regulated two-phase fermentation. Efficient cell growth and pyruvate production were reached separately by switching cultivation temperature. The results indicated that the genetic switch for controlling the noxE gene accurate expression was an effective strategy for improving pyruvate production.     

Engineering a <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> biosynthesis pathway into <Emphasis Type="Italic">Escherichia coli</Emphasis> for high yield triglyceride production

Background

Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.

Results

The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lpp β genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD_600nm of 80 and a product titer of 722.1 mg TAG L^-1 at the end of the process.

Conclusions

This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.

     

Long-term adaptation of <Emphasis Type="Italic">Escherichia coli</Emphasis> to methanogenic co-culture enhanced succinate production from crude glycerol

Escherichia coli can hardly grow anaerobically on glycerol without exogenous electron acceptor. The formate-consuming methanogen Methanobacterium formicicum plays a role as a living electron acceptor in glycerol fermentation of E. coli. Wild-type and mutant E. coli strains were screened for succinate production using glycerol in a co-culture with M. formicicum. Subsequently, E. coli was adapted to glycerol fermentation over 39 rounds (273 days) by successive co-culture with M. formicicum. The adapted E. coli (19.9 mM) produced twice as much succinate as non-adapted E. coli (9.7 mM) and 62% more methane. This study demonstrated improved succinate production from waste glycerol using an adapted wild-type strain of E. coli with wild-type M. formicicum, which is more useful than genetically modified strains. Crude glycerol, an economical feedstock, was used for the cultivation. Furthermore, the increase in methane production by M. formicicum during co-culture with adapted E. coli illustrated the possibility of energy-saving effects for the fermentation process.     

Sequence analysis of <Emphasis Type="Italic">KmXYL1</Emphasis> genes and verification of thermotolerant enzymatic activities of xylose reductase from four <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> strains

Kluyveromyces marxianus has the capability of producing xylitol from xylose because of the endogenous xylose reductase (KmXYL1) gene. In this study, we cloned KmXYL1 genes and compared amino acid sequences of xylose reductase (XR) from four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555). Four K. marxianus strains showed high homologies (99%) of amino acid sequences with those from other reported K. marxianus strains and around 60% homologies with that from Scheffersomyces stipitis. For XR enzymatic activities, four K. marxianus strains exhibited thermostable XR activities up to 45°C and K. marxianus KCTC 7001 showed the highest XR activity. When reaction temperatures were increased from 30 to 45°C, NADH-dependent XR activity from K. marxianus KCTC 7001 was highly increased (46%). When xylitol fermentations were performed at 30 or 45°C, four K. marxianus strains showed very poor xylitol production capabilities regardless fermentation temperatures. Xylitol productions from four K. marxianus strains might be limited because of low xylose uptake rate or cell growth although they have high thermostable XR activities.     

Evaluation and application of constitutive promoters for cutinase production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.     

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background

Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.

Results

In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.

Conclusion

Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.