Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum.

Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67 % over the parental strain (~12.6 g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4 % more butanol (18.2 ± 1.3 vs. 12.6 ± 0.2 g/L) with a 90 % higher productivity (0.38 ± 0.03 vs. 0.20 ± 0.02 g/L h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications.

Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production

In the transition to the post-petroleum economy, there is a growing demand for novel enzymes with high process performances to replace traditional chemistry with a more 'green' approach. To date, microorganisms encompass the richest source of industrial biocatalysts, but the Earth-living microbiota remains largely untapped by using traditional isolation and cultivation methods. Metagenomics, which is culture independent, represents a powerful tool for discovering novel enzymes from unculturable microorganisms. Herein, we summarize the variety of approaches adopted for mining environmental DNA and, based on a systematic literature review, we provide a comprehensive list of 332 industrially relevant enzymes discovered from metagenomes within the last three years.

Metagenomics: novel enzymes from non-culturable microbes.

Presently, several researchers are increasingly focusing on producing butanol as the next-generation fuel by acetone-butanol-ethanol (ABE) fermentation. Butanol has many superior characteristics compared to other biofuels, such as ethanol. However, its production by ABE fermentation faces the challenges of low productivity and yield because of product inhibition and heterofermentation, respectively, and thereby, high costs. Until date, molecular biological techniques and fermentation engineering methods have been applied for high butanol production. Although glucose remains the substrate of choice since traditional research, it is now necessary to substitute glucose derived from edible starch to other substrates from low-cost feedstock, such as agricultural residue. In addition, ABE-producing clostridia cannot directly produce butanol from lignocelluloses. Therefore, recent research is focusing on pretreatment and enzymatic saccharification of the complex molecules derived from agricultural residue for use as feedstock in butanol production. This article reviews traditional research, including the metabolism patterns and characteristics of ABE-producing clostridia. Furthermore, this article describes developments in ABE fermentation with respect to the establishment of highly efficient butanol production processes, such as batch, fed-batch, and continuous cultures, with the introduction of butanol removal, as well as butanol production from lignocellulosic biomasses or alternative substrates to sugars.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/elsc.201200128

Recent advances and future prospects for increased butanol production by acetone‐butanol‐ethanol fermentation

The renewed interests in clostridial acetone-butanol-ethanol (ABE) fermentation as a next-generation biofuel source led to significantly intensified research in the past few years. This mini-review focuses on the current status of metabolic engineering techniques available for the model organism of ABE fermentation, Clostridium acetobutylicum. A comprehensive survey of various application examples covers two general issues related to both basic and applied research questions: (i) how to improve biofuel production and (ii) what information can be deduced from respective genotype/phenotype manipulations. Recently developed strategies to engineer C. acetobutylicum are summarized including the current portfolio of altered gene expression methodologies, as well as systematic (rational) and explorative (combinatorial) metabolic engineering approaches.

Application of new metabolic engineering tools for Clostridium acetobutylicum.

The toxicity of n-butanol in microbial fermentations limits its formation. The stress response of Clostridium acetobutylicum involves various stress proteins and therefore, over-expression of genes encoding stress proteins constitutes an option to improve solvent tolerance. Over-expression of groESL, grpE and htpG, significantly improved butanol tolerance of C. acetobutylicum. Whereas the wild type and vector control strain did not survive 2 % (v/v) butanol for 2 h, the recombinant strains showed 45 % (groESL), 25 % (grpE) and 56 % (htpG), respectively, of the initial c.f.u. after 2 h of butanol exposure. As previously, over-expression of groESL led to higher butanol production rates, but the novel strains over-expressing grpE or htpG produced only 51 and 68 %, respectively, of the wild type butanol concentrations after 72 h clearly differentiating butanol tolerance and production. Not only butanol tolerance but also the adaptation to butanol in successive stress experiments was significantly facilitated by increased levels of GroESL, GrpE and HtpG. Re-transformation and sequence analyses of the plasmids confirmed that not the plasmids, but the host cells evolved to a more robust phenotype.

Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress

Two genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87 kDa) and GH36bt enzyme Agal2 (57 kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3 U/mg at 80 °C, pH 6.5, and Agal2 225.4 U/mg at 60–70 °C, pH 6.5. Half-lives of 14 h (Agal1) and 39 h (Agal2) were obtained at 50 °C, and Agal1 showed half-lives of 4 and 2 h at 70 and 80 °C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-d-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides.

Characterization of two novel heat-active α-galactosidases from thermophilic bacteria.

Exploiting enzymes for industrial purposes often requires engineering of these enzymes to adapt them to the industrial requirements. In order to meet industrial demands, we improved the thermostability of endoglucanase Cel7B from Hypocrea pseudokoningii ( Hp Cel7B), which was heterologously expressed in the yeast Pichia pastoris . Random mutants showing higher activity at elevated temperature have been selected and sequenced. In addition a model structure of our target enzyme was compared to structures of homologous but more thermostabile endoglucanases. This comparison pointed out several potential hot spots that were recognized as important for thermostability. The most promising mutations from both rational and non-rational approaches were randomly recombined by gene synthesis to evaluate potential additive effects for thermostability. This recombination library yielded a number of improved variants, of which the best ones were sequenced and characterized. Compared to the starting variant, recombination mutants showed up to 10 °C higher melting temperatures and can be used at higher temperatures than the natural enzyme.

Thermostability improvement of endoglucanase Cel7B from Hypocrea pseudokoningii

From a biogas reactor metagenome an ORF (bp_cel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BP_Cel9A exhibited ≤ 55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BP_Cel9A hydrolyzed the beta-1,3-1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (> 1%). In contrast to BP_Cel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BP_Cel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg2+ may adopt the function of Ca2+. BP_Cel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.

https://link.springer.com/content/pdf/10.1007%2Fs10930-018-9787-5.pdf

Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome.

Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in the Azores. Sequence comparison revealed that both genes had high similarities to genes from Fervidobacterium nodosum Rt17-B1. The two proteins showed sequence similarities of 62% to each other and belong to the glycoside hydrolase (GH) family 16. For biochemical characterization, both laminarinases were heterologously produced in Escherichia coli and purified to homogeneity. FLamA and FLamB exhibited similar properties and both showed highest activity towards laminarin at 90 °C and pH 6.5. The two enzymes were thermostable but differed in their half-life at 80 °C with 5 h and 1 h for FLamA and FLamB, respectively. In contrast to other laminarinases, both enzymes prefer β-1,3-glucans and mixed-linked glucans as substrates. However, FLamA and FLamB differ in their catalytic efficiency towards laminarin. Structure predictions were made and showed minor differences particularly in a kink adjacent to the active site cleft. The high specific activities and resistance to elevated temperatures and various additives make both enzymes suitable candidates for application in biomass conversion.

/pdf/comparative-analysis-and-biochemical-characterization-of-two-5azprzrafi.pdf

Georg Schirrmacher

Papers

Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress

Characterization of two novel heat-active α-galactosidases from thermophilic bacteria.

Thermostability improvement of endoglucanase Cel7B from Hypocrea pseudokoningii

Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome.

Comparative analysis and biochemical characterization of two endo-β-1,3-glucanases from the thermophilic bacterium fervidobacterium sp.