Thermophilic Geobacillus Thermoglucosidasius Research Articles

Metabolic engineering of Geobacillus thermoglucosidasius for polymer-grade lactic acid production at high temperature

The production and application of biodegradable polylactic acid are still severely hindered by the cost of its polymer-grade lactic acid monomers. High-temperature biomanufacturing has emerged as an increasingly attractive approach to enable low-cost and high-efficiency bulk chemical production. In this study, thermophilic Geobacillus thermoglucosidasius was reprogrammed to obtain optically pure l-lactic acid- and d-lactic acid-producing strains, G. thermoglucosidasius GTD17 and GTD7, by using rational metabolic engineering strategies including pathway construction, by-product elimination, and production enhancing. Moreover, semi-rational adaptive evolution was carried out to further improve their lactic acid synthesis performance. The final strains GTD17-55 and GTD7-144 produce 151.1 g/L of l-lactic acid and 153.1 g/L of d-lactic acid at 60 °C, respectively. In consideration of the high temperature, productive performance of these strains is superior compared to the state-of-the-art industrial strains. This study lays the foundation for the low-cost and efficient production of biodegradable plastic polylactic acid.

Optimization of consolidated bioprocessing by response surface methodology in the conversion of corn stover to bioethanol by thermophilic Geobacillus thermoglucosidasius.

The swift depletion of fossil fuels and their associated environment and economic impact has led the world to explore the sustainable alternate fuels. Amidst the available alternatives lignocellulosic bioethanol provides the edge over the exhausting fossil fuels. In this current study, Response surface methodology, a mathematical and statistical tool was used to optimize the fermentation conditions in consolidated bioprocessing of corn stover by Geobacillus thermoglucosidasius. The impact of inoculum concentration, temperature, pH, agitation speed and time in bioethanol fermentation were screened with Plackett-Burman design and it was farther optimized with central composite design. The analysis by PBD confirmed the significant impact of fermentation time, inoculum concentration, and temperature of the fermentation process. Further, it was optimized with CCD. This showed that 15% v/v of Inoculum concentration, 50°C of temperature and fermentation time of 72h increased the bioethanol concentration to a maximum of 9.04g/L with 0.45g/g significant yield and a conversion efficiency of 88%. Thus, the CCD showed a satisfactory result in consolidated bioprocessing of bioethanol from corn stover. Thus, in the future, this approach of optimization will yield a good base for consistent production of bioethanol.

Effect of Cultural Conditions on Protease Production by a Thermophilic Geobacillus thermoglucosidasius SKF4 Isolated from Sungai Klah Hot Spring Park, Malaysia.

Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 °C of temperature, pH 7 to 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.

Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production.

SummaryThe potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l−1). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribC Gtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpN Gtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l−1 after 12‐h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high‐temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.

Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955

BackgroundThe thermophile Geobacillus thermoglucosidasius has considerable attraction as a chassis for the production of chemicals and fuels. It utilises a wide range of sugars and oligosaccharides typical of those derived from lignocellulose and grows at elevated temperatures. The latter improves the rate of feed conversion, reduces fermentation cooling costs and minimises the risks of contamination. Full exploitation of its potential has been hindered by a dearth of effective gene tools.ResultsHere we designed and tested a collection of vectors (pMTL60000 series) in G. thermoglucosidasius NCIMB 11955 equivalent to the widely used clostridial pMTL80000 modular plasmid series. By combining a temperature-sensitive replicon and a heterologous pyrE gene from Geobacillus kaustophilus as a counter-selection marker, a highly effective and rapid gene knock-out/knock-in system was established. Its use required the initial creation of uracil auxotroph through deletion of pyrE using allele-coupled exchange (ACE) and selection for resistance to 5-fluoroorotic acid. The turnaround time for the construction of further mutants in this pyrE minus strain was typically 5 days. Following the creation of the desired mutant, the pyrE allele was restored to wild type, within 3 days, using ACE and selection for uracil prototrophy. Concomitant with this process, cargo DNA (pheB) could be readily integrated at the pyrE locus. The system’s utility was demonstrated through the generation in just 30 days of three independently engineered strains equivalent to a previously constructed ethanol production strain, TM242. This involved the creation of two in-frame deletions (ldh and pfl) and the replacement of a promoter region of a third gene (pdh) with an up-regulated variant. In no case did the production of ethanol match that of TM242. Genome sequencing of the parental strain, TM242, and constructed mutant derivatives suggested that NCIMB 11955 is prone to the emergence of random mutations which can dramatically affect phenotype.ConclusionsThe procedures and principles developed for clostridia, based on the use of pyrE alleles and ACE, may be readily deployed in G. thermoglucosidasius. Marker-less, in-frame deletion mutants can be rapidly generated in 5 days. However, ancillary mutations frequently arise, which can influence phenotype. This observation emphasises the need for improved screening and selection procedures at each step of the engineering processes, based on the generation of multiple, independent strains and whole-genome sequencing.

KINETIC EVALUATION OF ETHANOL-TOLERANT THERMOPHILE Geobacillus thermoglucosidasius M10EXG FOR ETHANOL PRODUCTION

Thermophiles are challenging to be studied for ethanol production using agricultural waste containing lignocellulosic materials rich in hexose and pentose. These bacteria have many advantages such as utilizing a wide range of substrates, including pentose (C5) and hexose (C6). In ethanol production, it is important to use ethanol tolerant strain capable in converting lignocellulosic hydrolysate. This study was aimed to investigate the growth profile of ethanol-tolerant thermophile Geobacillus thermoglucosidasius M10EXG using a defined growth medium consisted of single carbon glucose (TGTV), xylose (TXTV), and a mixture of glucose and xylose (TGXTV), together with the effect of yeast extract addition<br />to the media. The experiments were conducted at the School of Biotechnology and Biomolecular Sciences of The University of New South Wales, Australia on a shake flask fermentation at 60°C in duplicate experiment. Cultures were sampled every two hours and analised for their kinetic parameters including the maximum specific growth rate (µmax), biomass yield (Yx/s), ethanol and by-product yields (acetate and L-lactate) (Yp/s), and the doubling time (Td). Results showed that this strain was capable of growing on minimal medium containing glucose or xylose as a single carbon source. This strain utilized glucose and xylose simultaneously (co-fermentation), although there was glucose repression of xylose at relatively low glucose concentration (0.5% w/v), particularly when yeast extract (0.2% w/v) was added to the medium. The highest biomass yield was obtained at 0.5 g l-1 on glucose medium; the yield increased when yeast extract was added (at 0.59 g l-1). The highest specific growth rate of 0.25 was obtained in the phase I growth when the strain was grown on a mixture of glucose and xylose (0.5% : 0.5% w/v) medium. Diauxic growth was shown on the mixture of glucose, xylose, and yeast extract. The strain produced low level of ethanol (0.1 g l-1), as well as low level (0.2 g l-1) of by-products (L-lactate and acetate) after 15 hours. The results suggests its potential application for fermenting lignocellulosic agricultural wastes for ethanol production.

KINETIC EVALUATION OF ETHANOL-TOLERANT THERMOPHILE Geobacillus thermoglucosidasius M10EXG FOR ETHANOL PRODUCTION

Thermophiles are challenging to be studied for ethanol production using agricultural waste containing lignocellulosic materials rich in hexose and pentose. These bacteria have many advantages such as utilizing a wide range of substrates, including pentose (C5) and hexose (C6). In ethanol production, it is important to use ethanol tolerant strain capable in converting lignocellulosic hydrolysate. This study was aimed to investigate the growth profile of ethanol-tolerant thermophile Geobacillus thermoglucosidasius M10EXG using a defined growth medium consisted of single carbon glucose (TGTV), xylose (TXTV), and a mixture of glucose and xylose (TGXTV), together with the effect of yeast extract addition<br />to the media. The experiments were conducted at the School of Biotechnology and Biomolecular Sciences of The University of New South Wales, Australia on a shake flask fermentation at 60°C in duplicate experiment. Cultures were sampled every two hours and analised for their kinetic parameters including the maximum specific growth rate (µmax), biomass yield (Yx/s), ethanol and by-product yields (acetate and L-lactate) (Yp/s), and the doubling time (Td). Results showed that this strain was capable of growing on minimal medium containing glucose or xylose as a single carbon source. This strain utilized glucose and xylose simultaneously (co-fermentation), although there was glucose repression of xylose at relatively low glucose concentration (0.5% w/v), particularly when yeast extract (0.2% w/v) was added to the medium. The highest biomass yield was obtained at 0.5 g l-1 on glucose medium; the yield increased when yeast extract was added (at 0.59 g l-1). The highest specific growth rate of 0.25 was obtained in the phase I growth when the strain was grown on a mixture of glucose and xylose (0.5% : 0.5% w/v) medium. Diauxic growth was shown on the mixture of glucose, xylose, and yeast extract. The strain produced low level of ethanol (0.1 g l-1), as well as low level (0.2 g l-1) of by-products (L-lactate and acetate) after 15 hours. The results suggests its potential application for fermenting lignocellulosic agricultural wastes for ethanol production.

Complete Genome Sequence of Geobacillus thermoglucosidasius NCIMB 11955, the Progenitor of a Bioethanol Production Strain.

The industrially important thermophile Geobacillus thermoglucosidasius has the potential to produce chemicals and fuels from biomass-derived sugar feedstocks. Here, we present the genome sequence of strain NCIMB 11955, the progenitor of an ethanologenic industrial strain, revealing 11 single-nucleotide polymorphisms and 2 indels compared to strain DSM 2542 and two novel plasmids.

Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production

The ability to grow at high temperatures makes thermophiles attractive for many fermentation processes. In this work, we used evolutionary engineering to increase ethanol production in the thermophile Geobacillus thermoglucosidasius. This bacterium is a facultative anaerobe, grows at an optimal temperature of 60°C, and can ferment diverse carbohydrates. However, it natively performs mixed-acid fermentation. To improve ethanol productivity, we first eliminated lactate and formate production in two strains of G. thermoglucosidasius, 95A1 and C56-YS93. These deletion strains were generated by selection on spectinomycin, which represents, to the best of our knowledge, the first time this antibiotic has been shown to work with thermophiles. Both knockout strains, however, were unable to grow under microaerobic conditions. We were able to recover growth in G. thermoglucosidasius 95A1 by serial adaptation in the presence of acetic acid. The evolved 95A1 strain was able to efficiently produce ethanol during growth on glucose or cellobiose. Genome sequencing identified loss-of-function mutations in adenine phosphoribosyltransferase (aprt) and the stage III sporulation protein AA (spoIIIAA). Disruption of both genes improved ethanol production in the unadapted strains: however, the increase was significant only when aprt was deleted. In conclusion, we were able to engineer a strain of G. thermoglucosidasius to efficiently produce ethanol from glucose and cellobiose using a combination of metabolic engineering and evolutionary strategies. This work further establishes this thermophile as a platform organism for fuel and chemical production. Biotechnol. Bioeng. 2016;113: 2156-2167. © 2016 Wiley Periodicals, Inc.

Genome sequence of Geobacillus thermoglucosidasius DSM2542, a platform hosts for biotechnological applications with industrial potential

Thermophilic Geobacillus thermoglucosidasius could ferment a wide range of substrates with low nutrient requirements for growth. Here, the first released the complete genome sequence of G. thermoglucosidasius DSM2542 may facilitate the design of rational strategies for further strain improvements and provide information for exploring industrially interesting enzymes with thermotolerant properties.

Identification and characterization of a novel Geobacillus thermoglucosidasius bacteriophage, GVE3.

The study of extremophilic phages may reveal new phage families as well as different mechanisms of infection, propagation and lysis to those found in phages from temperate environments. We describe a novel siphovirus, GVE3, which infects the thermophile Geobacillus thermoglucosidasius. The genome size is 141,298 bp (G+C 29.6%), making it the largest Geobacillus spp-infecting phage known. GVE3 appears to be most closely related to the recently described Bacillus anthracis phage vB_BanS_Tsamsa, rather than Geobacillus-infecting phages described thus far. Tetranucleotide usage deviation analysis supports this relationship, showing that the GVE3 genome sequence correlates best with B. anthracis and Bacillus cereus genome sequences, rather than Geobacillus spp genome sequences.

Global sensitivity analysis and meta-modeling of an ethanol production process

Traditional ethanol fermentation becomes inhibitory to microbial growth at ethanol concentrations that depend on the producer organism, leading to reduced ethanol productivity. Continuous ethanol removal from the fermenter could increase productivity and potentially reduce the cost of product recovery. In this work, continuous ethanol removal via in situ gas stripping in a stirred tank reactor has been investigated as a means of reducing growth inhibition and improving productivity. A dynamic mathematical model that couples ethanol fermentation with gas stripping has been developed. This has been linked to a flash separation model to represent the initial steps of product recovery. Global sensitivity analysis was used to reduce the number of uncertain parameters, the values of which were estimated with satisfactory accuracy using experimental data for ethanol production from a metabolically engineered strain of the thermophile Geobacillus thermoglucosidasius growing on cellobiose. Simulation results show that continuous ethanol fermentation with product removal by gas stripping is feasible, with the associated energy requirement, costs of gas compression and fermenter agitation being a function of the stripping gas flow rate. Finally, the conditions under which gas stripping is a practical product recovery method were established.

Isobutanol production at elevated temperatures in thermophilic Geobacillus thermoglucosidasius

The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50°C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3g/l of isobutanol was produced from glucose and 0.6g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48h at 50°C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature.

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75 × 10(5) M(-1) s(-1) at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35 ± 0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.