Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity.

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd-EtfAB (BCD) complex was overexpressed, the resulting strain PRH-B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45-0.51 g/L/h and 0.46-0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin-NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.

Sub-MIC streptomycin and tetracycline enhanced Staphylococcus aureus Guangzhou-SAU749 biofilm formation, an in-depth study on transcriptomics.

Staphylococcus aureus is a major human pathogen, a potential "Super-bug" and a typical biofilm forming bacteria. With usage of large amount of antibiotics, the residual antibiotics in clinical settings further complicate the colonization, pathogenesis and resistance of S. aureus. This study aimed at investigating the phenotypical and global gene expression changes on biofilm formation of a clinical S. aureus isolate treated under different types of antibiotics. Firstly, an isolate Guangzhou-SAU749 was selected from a large sale of previously identified S. aureus isolates, which exhibited weak biofilm formation in terms of biomass and viability. Secondly, 9 commonly prescribed antibiotics for S. aureus infections treatment, together with 10 concentrations ranging from 1/128 to 4 minimum inhibitory concentration (MIC) with 2-fold serial dilution, were used as different antibiotic stress conditions. Then, biofilm formation of S. aureus Guangzhou-SAU749 at different stages including 8 h, 16 h, 24 h, and 48 h, was tested by crystal violet and MTS assays. Thirdly, the whole genome of S. aureus Guangzhou-SAU749 was investigated by genome sequencing on PacBio platform. Fourthly, since enhancement of biofilm formation occurred when treated with 1/2 MIC tetracycline (TCY) and 1/4 MIC streptomycin (STR) since 5 h, the relevant biofilm samples were selected and subjected to RNA-seq and bioinformatics analysis. Last, expression of two component system (TCS) and biofilm associated genes in 4 h, 8 h, 16 h, 24 h, and 48 h sub-MIC TCY and STR treated biofilm samples were performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Although most antibiotics lowered the biomass and cell viability of Guangzhou-SAU749 biofilm at concentrations higher than MIC, certain antibiotics including TCY and STR promoted biofilm formation at sub-MICs. Additionally, upon genome sequencing, RNA-seq and RT-qPCR on biofilm samples treated with sub-MIC of TCY and STR at key time points, genes lytR, arlR, hssR, tagA, clfB, atlA and cidA related to TCS and biofilm formation were identified to contribute to the enhanced biofilm formation, providing a theoretical basis for further controlling on S. aureus biofilm formation.

Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT27 for biofuels production from sucrose and molasses.

BACKGROUND: Sucrose-rich sugarcane trash surpasses 28 million tons globally per year. Effective biorefinery systems could convert these biomasses to bioproducts, such as bioethanol from sugarcane sucrose in Brazil. Thermophilic microbes for biofuels have attracted great attention due to their higher fermentation temperature and wide substrate spectrum. However, few thermophiles using sucrose or molasses for biofuels production was reported. Thermoanaerobacterium aotearoense SCUT27 has been considered as an efficient ethanol producer, but it cannot directly utilize sucrose. In this study, various sucrose metabolic pathways were introduced and analyzed in Thermoanaerobaterium. RESULTS: The sucrose-6-phosphate hydrolase (scrB), which was from a screened strain Thermoanaerobacterium thermosaccharolyticum G3-1 was overexpressed in T. aotearoense SCUT27 and endowed this strain with the ability to utilize sucrose. In addition, overexpression of the sucrose-specific PTS system (scrA) from Clostridium acetobutylicum accelerated the sucrose transport. To strengthen the alcohols production and substrates metabolism, the redox-sensing transcriptional repressor (rex) in T. aotearoense was further knocked out. Moreover, with the gene arginine repressor (argR) deleted, the ethanologenic mutant P8S10 showed great inhibitors-tolerance and finally accumulated ~ 34 g/L ethanol (a yield of 0.39 g/g sugars) from pretreated cane molasses in 5 L tank by fed-batch fermentation. When introducing butanol synthetic pathway, 3.22 g/L butanol was produced by P8SB4 with a yield of 0.44 g alcohols/g sugars at 50℃. This study demonstrated the potential application of T. aotearoense SCUT27 for ethanol and butanol production from low cost cane molasses. CONCLUSIONS: Our work provided strategies for sucrose utilization in thermophiles and improved biofuels production as well as stress tolerances of T. aotearoense SCUT27, demonstrating the potential application of the strain for cost-effective biofuels production from sucrose-based feedstocks.

Screening ester-producing yeasts to fortify the brewing of rice-flavor Baijiu for enhanced aromas.

To enhance the aromas in Guangdong rice-flavor Baijiu, ester-producing yeast was selected to fortify Baijiu brewing. Among eight kinds of ester-producing yeasts selected, Saccharomyces cerevisiae CM15 (CM15) that showed both the stronger ability to utilize substrates to produce esters and the excellent tolerance to industrially relevant stress factors was chosen. When CM15 was synergistically fermented with six kinds of Kojis from distilleries of rice-flavor liquor in Guangdong, the enhanced total esters had happened to the liquors brewing with the fortified four kinds of Kojis, especially with Koji F. When Koji F was fortified with CM15, the resultant Baijiu showed a higher esters proportion and a lower higher alcohol ratio than that of Baijiu brewed only with Koji F, with the content of ethyl acetate and ethyl lactate increasing by 25% and 214%, respectively. This study suggested that CM15 can be used as a functional microorganism to fortify Baijiu brewing, which might also be suitable for other traditional fermented foods.

Characterization and Correlation of Microbiota and Higher Alcohols Based on Metagenomic and Metabolite Profiling during Rice-Flavor Baijiu Fermentation.

Higher alcohol, as an inevitable product of fermentation, plays an important role in the flavor and quality of Baijiu. However, the relationship between the complex microbial metabolism and the formation of higher alcohols in rice-flavor Baijiu was not clear. To investigate the relationship between microorganisms and higher alcohol production, two fermentation mashes inoculated with starters from Heyuan Jinhuangtian Liquor Co., Ltd. (Heyuan, China) as JM and Guangdong Changleshao Co., Ltd. (Meizhou, China) as CM, respectively, with significant differences in higher alcohol profiles during rice-flavor Baijiu fermentation were selected. In general, higher alcohols presented a rapid accumulation during the early fermentation stages, especially in JM, with higher and faster increases than those in CM. As for their precursors including amino acids, pyruvic acid and ketoacids, complex variations were observed during the fermentation. Metagenomic results indicated that Saccharomyces cerevisiae and Rhizopus microsporus were the microorganisms present throughout the brewing process in JM and CM, and the relative abundance of R. microsporus in JM was significantly higher than that in CM. The results of higher alcohol metabolism in JM may contribute to the regulation of higher alcohols in rice-flavor Baijiu.

Exploiting the Type I-B CRISPR Genome Editing System in Thermoanaerobacterium aotearoense SCUT27 and Engineering the Strain for Enhanced Ethanol Production.

Thermoanaerobacterium aotearoense strain SCUT27 is a potential industrial biofuel-producing strain because of its broad substrate spectrum, especially the ability to co-use glucose and xylose. The bottleneck hindering the development of strain SCUT27 is the lack of selective markers for polygene manipulation in this thermophilic bacterium. In this study, the endogenous type I-B clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system was developed for multiplex genome editing of strain SCUT27. The protospacer-adjacent motif was identified by in silico analysis and verified with orotidine-5'-phosphate decarboxylase (pyrF) or lactate dehydrogenase (ldh) as the editing target. The type I-B CRISPR/Cas system was functional in strain SCUT27 with 58.3% to 100% editing efficiency. A multiplex genome editing method based on thymidine kinase (tdk) as a negative selection marker was developed, and strain SCUT27/Δtdk/Δldh/ΔargR, in which ldh and the arginine repressor (argR) were knocked out successively, was successfully obtained. Strain SCUT27/Δtdk/Δldh/ΔargR exhibited prominent advantages over wild-type SCUT27 in ethanol production, with significantly improved ability to metabolize xylose. IMPORTANCE Thermophilic microbes have attracted great attention as potential candidates for production of biofuels and chemicals from lignocellulose because of their thermal tolerance and wide substrate spectra. The ability to edit multiple genes using the native type I-B CRISPR/Cas system would speed up engineering of Thermoanaerobacterium aotearoense strain SCUT27 for higher ethanol production from lignocellulosic hydrolysates. Here, we produced a mutant strain, T. aotearoense SCUT27/Δtdk/Δldh/ΔargR, using the native CRISPR/Cas system. The engineered strain showed satisfactory performance with improved ethanol productivity from various lignocellulosic hydrolysates. Our data lay the foundations for development of this thermophilic microbe into an excellent ethanol producer using lignocellulosic hydrolysates. The methods described here may also provide a reference to develop multigene editing methods for other microorganisms.

Engineering Thermoanaerobacterium aotearoense SCUT27 with argR knockout for enhanced ethanol production from lignocellulosic hydrolysates.

Although Thermoanaerobacterium aotearoense SCUT27 (SCUT27) could co-utilize glucose and xylose, the presence of glucose still repressed xylose catabolism. Arginine repressors (ArgRs) were involved in several key metabolic pathways and might be the global regulator. In SCUT27, three genes (V518_0585; V518_1870; V518_1864) were annotated as argR and only the deficiency of argR1864 could greatly improve the co-utilization of glucose and xylose, due to the enhanced activity of xylose isomerase, xylulokinase and the higher energy level. The metabolic flux of SCUT27/ΔargR1864 indicated that new carbon distribution had been re-established and the ethanol yield had increased by 82.95%, strains growth and acetate yield improved by ~35.91% without detectable lactate for the poor activity of lactate dehydrogenase. The improved concentration of ATP and NAD(H) in SCUT27/ΔargR1864 provided more energy to respond the stress, which enabled the mutant the better cell viability to utilize lignocellulosic hydrolysates for enhanced ethanol formation.

The redox-sensing transcriptional repressor Rex is important for regulating the products distribution in Thermoanaerobacterium aotearoense SCUT27.

The redox-sensing transcriptional repressor Rex (Rex) displayed diverse functions in different microbial species. Nowadays, only part function of rex has been verified in vitro and alcohol dehydrogenase gene (adhE) as the target of Rex has been widely reported. In this study, rex was knocked out in Thermoanaerobacterium aotearoense SCUT27 (GDMCC 60765) and the carbon metabolic distribution analysis was performed. Results showed that the ethanol yield (mol product/mol carbon) of SCUT27(Δrex) had increased by 75.00-90.91%, cell growth improved by 27.27-36.36%, and acetic acid and lactic acid decreased by 58.33-61.54% accompanied with the yield of hydrogen decreased by 46.15-58.35% within different carbon sources. The ability of sugar consumption of SCUT27(Δrex) had improved about 74.19-130.55% with the improvement of total ATP concentration and the cofactors NADH and NAD+ concentrations. In addition, the specific activities of alcohol dehydrogenase of SCUT27(Δrex) with NADH and NADPH as cofactors were improved by 119.26-140.28% and 35.66-47.69%, respectively. After ldh was further knocked out in SCUT27(Δrex), SCUT27(ΔldhΔrex) showed higher ethanol production and yield when various carbon resources were used as substrates (including glucose, xylose, glucose/xylose mixture and three kinds of lignocellulosic hydrolysates). This study confirms that Rex is an important regulator for determining products distribution in SCUT27 and deletion of rex and ldh is a promising strategy for enhanced ethanol production.

Engineered Thermoanaerobacterium aotearoense with nfnAB knockout for improved hydrogen production from lignocellulose hydrolysates.

BACKGROUND: As a renewable and clean energy carrier, the production of biohydrogen from low-value feedstock such as lignocellulose has increasingly garnered interest. The NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB) complex catalyzes electron transfer between reduced ferredoxin and NAD(P)+, which is critical for production of NAD(P)H-dependent products such as hydrogen and ethanol. In this study, the effects on end-product formation of deletion of nfnAB from Thermoanaerobacterium aotearoense SCUT27 were investigated. RESULTS: Compared with the parental strain, the NADH/NAD+ ratio in the ∆nfnAB mutant was increased. The concentration of hydrogen and ethanol produced increased by (41.1 ± 2.37)% (p < 0.01) and (13.24 ± 1.12)% (p < 0.01), respectively, while the lactic acid concentration decreased by (11.88 ± 0.96)% (p < 0.01) when the ∆nfnAB mutant used glucose as sole carbon source. No obvious inhibition effect was observed for either SCUT27 or SCUT27/∆nfnAB when six types of lignocellulose hydrolysate pretreated with dilute acid were used for hydrogen production. Notably, the SCUT27/∆nfnAB mutant produced 190.63-209.31 mmol/L hydrogen, with a yield of 1.66-1.77 mol/mol and productivity of 12.71-13.95 mmol/L h from nonsterilized rice straw and corn cob hydrolysates pretreated with dilute acid. CONCLUSIONS: The T. aotearoense SCUT27/∆nfnAB mutant showed higher hydrogen yield and productivity compared with those of the parental strain. Hence, we demonstrate that deletion of nfnAB from T. aotearoense SCUT27 is an effective approach to improve hydrogen production by redirecting the electron flux, and SCUT27/∆nfnAB is a promising candidate strain for efficient biohydrogen production from lignocellulosic hydrolysates.

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from undetoxified corncob acid hydrolysate.

Resistance to furan derivatives and phenolic compounds plays an important role in the use of lignocellulosic biomass for biological production of chemicals and fuels. This study confirmed that expression of short-chain dehydrogenase/reductase (SDR) from Clostridium beijerinckii NCIMB 8052 significantly improved the tolerance of C. tyrobutyricum to furfural due to the enhanced activity for furfural reduction. And on this basis, co-expression of SDR and heat shock chaperones GroESL could simultaneously enhance the tolerance of C. tyrobutyricum to furan derivatives and phenolic compounds, which were the main inhibitors presented in dilute-acid lignocellulosic hydrolysates. Consequently, the recombinant strain ATCC 25755/sdr+groESL exhibited good performance in butyric acid production with corncob acid hydrolysate as the substrate. Batch fermentation in bioreactor showed that the butyrate produced by ATCC 25755/sdr+groESL was 32.8 g/L, increased by 28.1% as compared with the wild-type strain. Meanwhile, the butyrate productivity increased from 0.19 g/L·h to 0.29 g/L·h.

Enhanced isopropanol and n-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.