菊糖芽孢乳杆菌DS2-18中试规模的D-乳酸发酵研究

研究了菊糖芽孢乳杆茵DS2的突变株DS2-18在中试规模的D-乳酸发酵.在容积为300L自控发酵罐中,DS2-18茵在合适的发酵条件下,即培养基组成(g/L):葡萄糖120,玉米浆8,蛋白胨6,豆粕水解液100,接种量8%(v/v),发酵温度40℃,以轻质碳酸钙作为中和剂调pH 5～6,发酵期间交替不通气和通气,发酵6...     

Draft genome sequence of Sporolactobacillus inulinus strain CASD, an efficient D-lactic acid-producing bacterium with high-concentration lactate tolerance capability

Sporolactobacillus inulinus CASD is an efficient D-lactic acid producer with high optical purity. Here we report for the first time the draft genome sequence of S. inulinus (2,930,096 bp). The large number of annotated two-component system genes makes it possible to explore the mechanism of extraordinary lactate tolerance of S. inulinus CASD.     

Biological characterization of D-lactate dehydrogenase responsible for high-yield production of D-phenyllactic acid in Sporolactobacillus inulinus

PLA (3-D-phenyllactic acid) is an ideal antimicrobial and immune regulatory compound present in honey and fermented foods. Sporolactobacillus inulinus is regarded as a potent D-PLA producer that reduces phenylpyruvate (PPA) with D-lactate dehydrogenases. In this study, PLA was produced by whole-cell bioconversion of S. inulinus ATCC 15538. Three genes encoding D-lactate dehydrogenase (d-ldh1, d-ldh2, and d-ldh3) were cloned and expressed in Escherichia coli BL21 (DE3), and their biochemical and structural properties were characterized. Consequently, a high concentration of pure D-PLA (47 mM) was produced with a high conversion yield of 88%. Among the three enzymes, D-LDH1 was responsible for the efficient conversion of PPA to PLA with kinetic parameters of Km (0.36 mM), k_cat (481.10 s⁻¹), and k_cat/Km (1336.39 mM⁻¹ s⁻¹). In silico structural analysis and site-directed mutagenesis revealed that the Ile307 in D-LDH1 is a key residue for excellent PPA reduction with low steric hindrance at the substrate entrance. This study highlights that S. inulinus ATCC 15538 is an excellent PLA producer, equipped with a highly specific and efficient D-LDH1 enzyme.     

Glucokinase contributes to glucose phosphorylation in d-lactic acid production by Sporolactobacillus inulinus Y2-8

Sporolactobacillus inulinus, a homofermentative lactic acid bacterium, is a species capable of efficient industrial d-lactic acid production from glucose. Glucose phosphorylation is the key step of glucose metabolism, and fine-tuned expression of which can improve d-lactic acid production. During growth on high-concentration glucose, a fast induction of high glucokinase (GLK) activity was observed, and paralleled the patterns of glucose consumption and d-lactic acid accumulation, while phosphoenolpyruvate phosphotransferase system (PTS) activity was completely repressed. The transmembrane proton gradient of 1.3–1.5 units was expected to generate a large proton motive force to the uptake of glucose. This suggests that the GLK pathway is the major route for glucose utilization, with the uptake of glucose through PTS-independent transport systems and phosphorylation of glucose by GLK in S. inulinus d-lactic acid production. The gene encoding GLK was cloned from S. inulinus and expressed in Escherichia coli. The amino acid sequence revealed significant similarity to GLK sequences from Bacillaceae. The recombinant GLK was purified and shown to be a homodimer with a subunit molecular mass of 34.5?kDa. Strikingly, it demonstrated an unusual broad substrate specificity, catalyzing phosphorylation of 2-deoxyglucose, mannitol, maltose, galactose and glucosamine, in addition to glucose. This report documented the key step concerning glucose phosphorylation of S. inulinus, which will help to understand the regulation of glucose metabolism and d-lactic acid production.     

Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling

Genome shuffling based on cross mating was used to improve the tolerance of the pentose-fermenting yeast Pichia stipitis towards hardwood spent sulphite liquor (HW SSL). Six UV-induced mutants of P. stipitis were used as the starting strains, and they were subjected to 4 rounds of genome shuffling. After each round, improved strains were selected based on their growth on HW SSL gradient plates. Mutant libraries were established after each round and these improved mutant strains served as the starting pool for the next round of shuffling. Apparent tolerance to HW SSL on the gradient plate increased progressively with each round of shuffling up to 4 rounds. Selected improved mutants were further tested for tolerance to liquid HW SSL. After 4 rounds of shuffling, 4 mutants, two from the third round (designated as GS301 and GS302) and two from the fourth round (designated as GS401 and GS402), were selected that could grow in 80% (v/v) HW SSL. GS301 and GS302 grew also in 85% (v/v) HW SSL. GS301 was viable in 90% (v/v) HW SSL, although no increase in cell number was seen. The P. stipitis wild type strain (WT) could not grow on HW SSL unless it was diluted to 65% (v/v) or lower. Genome-shuffled strains with improved tolerance to HW SSL retained their fermentation ability. Fermentation performance of GS301 and GS302, the 2 strains that exhibited the best tolerance to liquid HW SSL, was assessed in defined media and in HW SSL. Both strains utilized 4% (w/v) of xylose or glucose more efficiently and produced more ethanol than the WT. They also utilized 4% (w/v) of mannose or galactose and produced ethanol to the same extent as the WT. GS301 and GS302 were able to produce low levels of ethanol in undiluted HW SSL.     

Effect of lignocellulose-derived inhibitors on the growth and d-lactic acid production of Sporolactobacillus inulinus YBS1-5

Bioprocess and Biosystems Engineering - The impact of lignocellulose-derived inhibitors on the cell growth and d-lactic production of Sporolactobacillus inulinus YBS1-5 was investigated. At high...     

Improved production of propionic acid using genome shuffling

Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by‐products gene‐knockout strategies as a logical solution to increase yield. However, removing by‐product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well‐conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions.     

Sodium ions activated phosphofructokinase leading to enhanced d-lactic acid production by Sporolactobacillus inulinus using sodium hydroxide as a neutralizing agent

Sporolactobacillus inulinus is a superior d-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for d-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and d-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na⁺ was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO₃. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na⁺-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the d-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na⁺-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient d-lactic acid production with easy subsequent purification.

     

ARTP mutation and genome shuffling of ABE fermentation symbiotic system for improvement of butanol production

Butanol is an ideal renewable biofuel which possesses superior fuel properties. Previously, butanol-producing symbiotic system TSH06 was isolated in our lab, with microoxygen tolerance ability. To boost butanol yield for large-scale industrial production, TSH06 was used as parental strain and subjected to atmospheric and room temperature plasma (ARTP) and four rounds of genome shuffling (GS). ARTP mutant and GS strain were co-cultured with facultative anaerobic Bacillus cereus TSH2 to form a symbiotic system with microoxygen tolerance, which was then subjected to fermentation. Relative messenger RNA (mRNA) level of key enzyme gene was measured by real-time PCR. The highest butanol titer of TS4-30 reached 15.63 g/L, which was 34% higher than TSH06 (12.19 g/L). Compared with parental strain, mRNA of acid-forming gene in TS4-30 decreased in acidogenesis phase, while solvent-forming gene increased in solventogenesis phase. This gene expression pattern was consistent with high butanol yield and low acid level in TS4-30. In summary, symbiotic system TS4-30 was obtained with butanol titer improvement and microoxygen tolerance.

     

Genome shuffling enhanced L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we applied the genome shuffling to improve the glucose tolerance of Lactobacillus rhamnosus ATCC 11443 while simultaneously enhancing the L-lactic acid production. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for the recursive protoplast fusion. The positive colonies from library created by fusing the inactivated protoplasts were more likely to be screened on plates containing different concentrations of high glucose and 2% CaCO(3). Characterization of all mutants and wild-type strain in the shake flask indicated the compatibility of two optimal phenotypes of glucose tolerance and lactic acid enhancement. The lactic acid production, cell growth and glucose consumption of the best performing strain from the second round genome shuffled populations were 71.4%, 44.9% and 62.2% higher than those of the wild type at the initial glucose concentration of 150 g/l in the 16l bioreactor. Furthermore, the higher lactic acid concentrations were obtained when the initial glucose concentrations increased to 160 and 200 g/l in batch fermentation.     

Genome shuffling of Lactobacillus for improved acid tolerance

Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.     

Evolution of Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling

Improvement of pristinamycin production by Streptomyces pristinaespiralis was performed by using recursive protoplast fusion and selection for improved resistance to the product antibiotic in a genome shuffling format. A 100-mug/ml pristinamycin resistant recombinant, G 4-17, was obtained after four rounds of protoplast fusion, and its production of pristinamycin reached 0.89 g/l, which was increased by 89.4% and 145.9% in comparison with that of the highest parent strain M-156 and the original strain CGMCC 0957, respectively. The subculture experiments indicated that the hereditary character of high producing S. pristinaespiralis G 4-17 was stable. It is concluded that genome shuffling improves the production of pristinamycin by enhancing product-resistance in a stepwise manner. Pristinamycin fermentation experiments by recombinant G 4-17 were carried out in a 5-l fermentor, and its production of pristinamycin reached 0.90 g/l after 60 h of fermentation.     

Improvement of pristinamycin production by genome shuffling and medium optimization for Streptomyces pristinaespiralis

To isolate an improved pristinamycin producing strain of Streptomyces pristinaespiralis, the technique of Genome shuffling was used which resulted in a high-yield recombinant G 3-56 strain. Strain G 3-56 yielded 322 ± 17 mg/L of pristinamycin which was 11.4-fold higher than that of the initial strain and 3.7-fold higher than strain UN-78 which previously had the highest yield of pristinamycin. The genetic characteristics of the recombinant G 3-56 strain was stable as revealed by our subculture experiments. The optimal production medium was determined using the orthogonal matrix method. Under the optimal medium conditions, the maximum yield of pristinamycin was 412 mg/L with about 1.24-fold higher than the original medium.     

Erratum to: Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling

Applied Microbiology and Biotechnology -     

D-乳酸高产菌菊糖芽胞乳杆菌Y2-8磷酸果糖激酶基因在大肠杆菌中的克隆和表达

以D-乳酸高产菌菊糖芽胞乳杆菌Y2-8基因组DNA为模板,通过PCR扩增得到960 bp的磷酸果糖激酶基因(pfk)。氨基酸序列比对分析表明,该磷酸果糖激酶(PFK)与其他乳酸菌PFK具有保守的底物结合位点,但是其变构效应物结合位点存在差异。将pfk基因克隆到表达载体pSE380上,获得重组菌E-pSE-pfk。进一步通过诱导条件的优化,重组菌的PFK比酶活达到4.89 U/mg,是优化前的4.79倍。采用低温诱导策略有助于实现菊糖芽胞乳杆菌pfk基因在大肠杆菌中可溶性表达。     

Strain improvement of Lactobacillus delbrueckii using nitrous acid mutation for l-lactic acid production

Insertional mutagenesis is impractical in the mechanisms for protection against low pH, high solute concentration etc. due to the involvement of large number of loci and multiple genes. An attempt was made to improve Lactobacillus delbrueckii NCIM 2025 strain by classical mutation using nitrous acid. In the present investigation, classical mutation was proved to be successful and the selected mutants had improved qualities like increased lactic acid productivity, acid tolerance and sugar tolerance. Mutants showed better growth rate and lesser generation time than the wild type.     

Xylose‐fermenting Pichia stipitis by genome shuffling for improved ethanol production

Xylose fermentation is necessary for the bioconversion of lignocellulose to ethanol as fuel, but wild‐type Saccharomyces cerevisiae strains cannot fully metabolize xylose. Several efforts have been made to obtain microbial strains with enhanced xylose fermentation. However, xylose fermentation remains a serious challenge because of the complexity of lignocellulosic biomass hydrolysates. Genome shuffling has been widely used for the rapid improvement of industrially important microbial strains. After two rounds of genome shuffling, a genetically stable, high‐ethanol‐producing strain was obtained. Designated as TJ2‐3, this strain could ferment xylose and produce 1.5 times more ethanol than wild‐type Pichia stipitis after fermentation for 96 h. The acridine orange and propidium iodide uptake assays showed that the maintenance of yeast cell membrane integrity is important for ethanol fermentation. This study highlights the importance of genome shuffling in P. stipitis as an effective method for enhancing the productivity of industrial strains.     

酿酒酵母产D-乳酸重组菌的构建与发酵

采用基因工程方法对酿酒酵母进行代谢改造,使酵母产生乳酸代谢途径。将来源于L.mesenteroides和E.coli的D-乳酸脱氢酶基因,分别插入带有G418抗性的酵母穿梭质粒p YX212-kan MX上,电转化酵母,得到2株生产D-乳酸的酿酒酵母重组菌S.cerevisiae WE1510和S.cerevisiae WB1186。进一步摇瓶发酵试验表明:重组菌S.cerevisiae WB1186在YEPD培养基、20 g/L糖、p H 5的条件下生长条件最好,并具有更好的产乳酸能力。经3 L发酵罐条件下验证,S.cerevisiae WB1186分批发酵96 h,最终乳酸积累量达到18.0 g/L;发酵条件为培养基YEPD,接种量10%,溶解氧(DO)30%,转速150 r/min,初始葡萄糖质量浓度10 g/L,控制pH 5.0,通气量3 L/min,OD600最大值转为厌氧发酵。     

Continuous production of lactic acid from molasses by free and immobilized Sporolactobacillus cellulosolvens

Both free and immobilized cells of Sporolactobacillus cellulosolvens, in continuous culture on molasses (50 g sugar 1^-1) at 40°C, had maximum lactic acid productivities of 0.03 and 0.06 mol l^-1 h, at dilution rates of 0.27 and 0.25 h^-1, respectively.S.S. Kanwar is with the Department of Biotechnology, Guru Nanak Dev University, Amritsar-143 005, India; B.S. Chadha is with the Department of Microbiology, Guru Nanak Dev University, Amritsar-143 005, India. H.K. Tewari and V.K. Sharma are with the Department of Microbiology, Punjab Agricultural University, Ludhiana-141 004, India.