Improvement of acetoin reductase activity enhances bacitracin production by Bacillus licheniformis

Bacitracin fermentation by Bacillus licheniformis in this work showed three characteristics: (1) the extracellular propionate, butyrate, acetoin and 2,3-butanediol accumulates under conditions of low dissolved oxygen (zero after 4 h cultivation), reaching a total content of approximately 11.1 g/L; (2) cell growth occurs quickly subsequent to cell autolysis and the second growth; and (3) there is a low content of 2,3-butanediol, a reduced product of acetoin catalyzed by acetoin reductase, in the culture process. In this study, addition of MnCl₂ (0.3 mg/L) to the production medium increased the acetoin reductase activity, redirected the NADH oxidation partly from the propionate- and butyrate-production pathways to the 2,3-butanediol synthesis pathway, reduced the intracellular NADH/NAD⁺ ratio, and facilitated cell growth, ultimately achieving a 11.6% increase in bacitracin production (1076 U/mL) versus the control. The results provide useful information regarding large-scale bacitracin production by B. licheniformis.     

The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7 g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639 g/(l h).     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

Compartmentalizing metabolic pathway in Candida glabrata for acetoin production

Acetoin, a valuable compound, has high potential as a biochemical building block. In this study, subcellular metabolic engineering was applied to engineer the mitochondrion of Candida glabrata for acetoin production. With the aid of mitochondrial targeting sequences, a heterologous acetoin pathway was targeted into the mitochondria to increase the enzyme concentrations and level of intermediate, followed by coupling with the mitochondrial pyruvate carrier (MPC) to increase the availability of mitochondrial pyruvate. As a result, the strain comprising the combination of the mitochondrial pathway and MPC could yield approximately 3.26 g/L of acetoin, which was about 59.8% higher than that produced by the cytoplasmic pathway. These results provided a new insight into the metabolic engineering of C. glabrata for acetoin production, and offered a potential platform to improve the performance of engineered pathways.     

Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis

When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD⁺, and a high titer of 371 mM (32 g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361 mM (32 g/L) R-BDO with a yield of 81% or 365 mM (33 g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production.     

Temperature-dependent acetoin production by Pyrococcus furiosus is catalyzed by a biosynthetic acetolactate synthase and its deletion improves ethanol production

The hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally near 100 °C by fermenting sugars to acetate, carbon dioxide and molecular hydrogen as the major end products. The organism has recently been exploited to produce biofuels using a temperature-dependent metabolic switch using genes from microorganisms that grow near 70 °C. However, little is known about its metabolism at the lower temperatures. We show here that P. furiosus produces acetoin (3-hydroxybutanone) as a major product at temperatures below 80 °C. A novel type of acetolactate synthase (ALS), which is involved in branched-chain amino acid biosynthesis, is responsible and deletion of the als gene abolishes acetoin production. Accordingly, deletion of als in a strain of P. furiosus containing a novel pathway for ethanol production significantly improved the yield of ethanol. These results also demonstrate that P. furiosus is a potential platform for the biological production of acetoin at temperatures in the 70–80 °C range.     

Metabolic engineering of Bacillus sp. for diacetyl production

Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe³⁺ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.     

Effect of gene knockouts of l-tryptophan uptake system on the production of l-tryptophan in Escherichia coli

Uptake of l-tryptophan in Escherichia coli was carried out by three distinct permeases, Mtr, TnaB, and AroP, respectively. In the present study, the three genes of l-tryptophan uptake system were knocked out from an l-tryptophan-producing strain of E. coli, respectively. The knockout mutants all showed lower l-tryptophan uptake activities and higher l-tryptophan production than their parent. Among the three genes, the knockout of mtr was most critical for both l-tryptophan uptake and l-tryptophan production. The uptake activity of l-tryptophan of the mtr mutant was 1.5 nmol min^?1 (mg dry weight)^?1, which was decreased by 48% when compared to that of the parent; the production of l-tryptophan of the mtr mutant was 14.7 g/l, which was increased by 34% when compared to that of the parent. Furthermore, the physiological and fermentation characteristics caused by gene knockouts were also analyzed.     

Establishment of a novel gene expression method,BICES (biomass-inducible chromosome-based expression system), and its application to the production of 2,3-butanediol and acetoin

In this study, we describe a novel method for producing valuable chemicals from glucose and xylose in Escherichia coli. The notable features in our method are avoidance of plasmids and expensive inducers for foreign gene expression to reduce production costs; foreign genes are knocked into the chromosome, and their expression is induced with xylose that is present in most biomass feedstock. As loci for the gene knock-in, lacZYA and some pseudogenes are chosen to minimize unexpected effects of the knock-in on cell physiology. The promoter of xylF is inducible with xylose and is combined with the T7 RNA polymerase–T7 promoter system to ensure strong gene expression. This expression system was named BICES (biomass-inducible chromosome-based expression system). As examples of BICES application, 2,3-butanediol and acetoin were successfully produced from glucose and xylose, and the maximal concentrations reached 54 g L⁻¹ [99.6% in (R,S)-form] and 31 g L⁻¹, respectively. 2,3-Butanediol and acetoin are industrially important chemicals that are, at present, produced primarily through petrochemical processes. To demonstrate usability of BICES in practical situations, we produced these chemicals from a saccharified cedar solution. From these results, we can conclude that BICES is suitable for practical production of valuable chemicals from biomass.     

高产3-羟基丁酮解淀粉芽孢杆菌的选育及发酵优化

3-羟基丁酮(Acetoin)作为一种重要的食用香料和平台化合物被广泛应用于医药、食品等领域。为改善解淀粉芽孢杆菌Bacillus amyloliquefaciens的3-羟基丁酮生产能力,采用常压室温等离子体(ARTP)和~(60)Coγ射线进行复合诱变,以3-羟基丁酮产量为分析指标,筛选获得最优突变株B.amyloliquefaciens H-5,3-羟基丁酮产量为68.2 g/L。为进一步实现3-羟基丁酮的高效生产,对此突变株进行5 L发酵罐水平的培养条件优化,并于30 L发酵罐上进行放大培养,最终3-羟基丁酮产量达85.2 g/L,较出发菌株B.amyloliquefaciens FMME088提高了26.8%。上述研究结果表明,ARTP和~(60)Coγ射线复合诱变能够有效获得高产菌株,该突变株具有较高的工业化微生物发酵生产3-羟基丁酮的潜能。     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

Efficient screening a high glutathione-content mutant of Saccharomyces cerevisiae by flow cytometry

Screening for the high glutathione-content microorganisms is an important technique for industrial production of glutathione. In this study, the intracellular glutathione-content was investigated to correlate better with the fluorescence emission at around 490 nm in the yeast. Therefore, we developed an efficient flow cytometry method to screen for the higher glutathione-content mutant of Saccharomyces cerevisiae by measuring the intensity of fluorescence emitted from the cell. The library of mutants was generated by ethyl methanesulfonate mutagenesis and mutant G-143 was isolated which produced 35% higher glutathione than the parent strain in the flask culture, and the glutathione yield was 37% increased when G-143 was scaled up to a 5 L fermentor for glutathione was the intracellular product.     

Enhanced acetone/butanol/ethanol production by Clostridium beijerinckii IB4 using pH control strategy

PH is an essential factor for acetone/butanol/ethanol (ABE) production using Clostridium spp. In this study, batch fermentations by Clostridium beijerinckii IB4 at various pH values ranging from 4.9 to 6.0 were examined. At pH 5.5, the ABE production was dominant and maximum ABE concentration of 24.6 g/L (15.7 g/L of butanol, 8.63 g/L of acetone and 0.32 g/L of ethanol) was obtained with the consumption of 60 g/L of glucose within 36 h. However, in the control (without pH control), an ABE concentration of 14.1 g/L (11.0 g/L of butanol, 3.01 g/L of acetone and 0.16 g/L of ethanol) was achieved with the consumption of 41 g/L of glucose within 40 h. A considerable improvement in the productivity of up to 93.8% was recorded at controlled pH in comparison to the process without pH control. To better understand the influence of pH on butanol production, the reducing power capability and NADH-dependent butanol dehydrogenase activity were investigated, both of which were significantly improved at pH 5.5. Thus, the pH control technique is a convenient and efficient method for high-intensity ABE production.     

High-level production of erythritol by mutants of osmophilic Moniliella sp.

An erythritol-producing osmophilic yeast-like fungus, Moniliella sp. 440, was isolated from honey and then successively mutated with iterative rounds of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment and selection. Six generations of mutants, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected for and produced erythritol at 151.0, 157.2, 177.8, 191.4, 196.6, and 237.8 g/L, respectively, while the wild type strain produced 113.0 g/L erythritol in media containing 40% glucose and 1% yeast extract. The mutant cells were found to have a short rod-like shape, while the wild type cells have a long rod-like shape. The most efficient erythritol producer, N61188-12, assimilated myo-inositol and weakly assimilated erythritol. However, the wild type strain did not assimilate myo-inositol and assimilated erythritol well. In 250-L and 2000-L pilot-scale fermentors, the erythritol production by N61188-12 was 151.4 g/L and 152.4 g/L, respectively. A simple fed-batch culture of strain N61188-12 in a 2000-L fermentor increased erythritol production to 189.4 g/L after 10 days fermentation.     

Enhancing production of prodigiosin from Serratia marcescens C3 by statistical experimental design and porous carrier addition strategy

Serratia marcescens C3 produces a natural red-pigment, prodigiosin, which exhibits immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. This work seeks to improve the production of prodigiosin by S. marcescens C3 using various strategies. Starch and peptone were identified as the optimized carbon and nitrogen sources for the production of prodigiosin, yielding a prodigiosin concentration of 2.3 g/L. This value was significantly increased to 6.7 g/L using a carbon/nitrogen ratio of 6/4 (starch/peptone = 16 g/L/10.67 g/L). To enhance prodigiosin production even further, a statistical experimental design methodology was utilized to optimize the composition of the culture medium that is utilized in the production of prodigiosin. Prodigiosin production of 7.07 g/L was achieved when the concentrations of two trace compounds, FeSO₄·4H₂O and MnSO₄·4H₂O, were optimized using the statistical experimental design methodology. Their optimal concentrations were 0.56 mM and 3.25 mM, respectively. Ultimately, the production of prodigiosin was increased from 2.3 g/L to 15.6 g/L, or by a factor of nearly seven by immobilizing microorganisms in 3% calcium alginate beads.     

Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution

This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38 U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55 °C and 50 °C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60 °C and apparent melting temperature of reBaxA50 were 9.74 min and 89.15 °C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

Unmarked insertional inactivation in the gfo gene improves growth and ethanol production by Zymomonas mobilis ZM4 in sucrose without formation of sorbitol as a by-product,but yields opposite effects in high glucose

Sorbitol, one of the main by-products of growth on high sucrose concentrations, is catalyzed by glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28) in Zymomonas mobilis, which decreases the ethanol yield. In this study, an unmarked gfo mutant from Z. mobilis ZM4 was constructed using a site-specific FLP recombinase, and growth and ethanol production were evaluated with or without the addition of sorbitol to the media. The inactivation of gfo had contrasting effects in different substrates, especially at high concentrations. The maximum specific growth rate (μ_m) and theoretical ethanol yield value (Y_m) increased from 0.065 h⁻¹ and 60.56% to 0.094 h⁻¹ and 83.87% in 342 g/L sucrose, respectively. Conversely, in 200 g/L glucose, gfo inactivation decreased μ_m and Y_m from 0.15 h⁻¹ and 89.85% to 0.10 h⁻¹ and 67.59%, respectively, and prolonged the lag period from 16 h to 40 h. The addition of sorbitol slightly accelerated growth and sucrose hydrolysis by the gfo mutant in 342 g/L sucrose; however, addition of sorbitol restored the μ_m and Y_m of the gfo mutant in 200 g/L glucose to 0.14 h⁻¹ and 82.50%, respectively. Inactivation of gfo had a small effect on fructose utilization, and a positive one on mixture of glucose and fructose similar to that on sucrose. These results provide further understanding of the osmoregulation mechanisms in Z. mobilis and may help to exploit the biotechnological applications of this industrially important bacterium.