Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives of Escherichia coli KO11

This study demonstrates a new approach to reduce the amount of fungal cellulase required for the conversion of cellulose into ethanol. Escherichia coli KO11, a biocatalyst developed for the fermentation of hemicellulose syrups, was used to produce recombinant endoglucanase as a co-product with ethanol. Seven different bacterial genes were expressed from plasmids in KO11. All produced cell-associated endoglucanase activity. KO11(pLOI1620) containing Erwinia chrysanthemi celZ (EGZ) produced the highest activity, 3,200 IU endoglucanase/L fermentation broth (assayed at pH 5.2 and 35 degrees C). Recombinant EGZ was solubilized from harvested cells by treatment with dilute sodium dodecyl sulfate (12.5 mg/ml, 10 min, 50 degrees C) and tested in fermentation experiments with commercial fungal cellulase (5 filter paper units/g cellulose) and purified cellulose (100 g/L). Using Klebsiella oxytoca P2 as the biocatalyst, fermentations supplemented with EGZ as a detergent-lysate of KO11(pLOI1620) produced 14%-24% more ethanol than control fermentations supplemented with a detergent-lysate of KO11(pUC18). These results demonstrate that recombinant bacterial endoglucanase can function with fungal cellulase to increase ethanol yield during the simultaneous saccharification and fermentation of cellulose. (c) 1997 Wiley & Sons, Inc. Biotechnol Bioeng 55: 547-555, 1997.     

Fermentative production of succinic acid from glucose and corn steep liquor byAnaerobiospirillum succiniciproducens

Anaerobiospirillum succiniciproducens requires expensive complex nitrogen sources such as yeast extract and polypeptone for its growth and succinic acid production. It was found thatA. succiniciproducens was able to grow in a minimal medium containing glucose when supplemented with corn steep liquor (CSL) as the sole complex nitrogen source. The concentration of CSL had a significant effect on the glucose consumption byA. succiniciproducens. When 10–15 g/L of CSL was supplemented, cells were grown to an OD₆₆₀ of 3.5 and produced 17.8 g/L succinic acid with 20 g/L glucose. These results are similar to those obtained by supplementing yeast extract and polypeptone, thereby suggesting that succinic acid can be produced more economically using glucose and CSL.     

Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a Limited Nutrient Medium

This study was designed to investigate the ability of Clostridium saccharoperbutylacetonicum N1-4 to produce butanol in a limited nutrient medium using mixtures of glucose and butyric acid as substrates. Specific combinations of glucose and butyric acid were found to influence the enhancement and retardation of butanol production as well as the reduction and modulation of the number of bacterial cells. Increasing the butyric acid concentration leads to the inhibition of bacterial growth, whereas the presence of (0?C5?g/L) butyric acid and (0?C10?g/L) glucose enhances the butanol production. The combination of 5?g/L butyric acid with 5 and 10?g/L of glucose was found to be the most suitable, but the use of glucose at concentrations greater than 10?g/L shifted the optimal butyric acid concentrations to 10 and 15?g/L for maximum butanol production signifying the requirement of a specific combination of glucose and butyric acid for enhanced butanol production in the fermentation process. C. saccharoperbutylacetonicum N1-4 demonstrated the ability to produce butanol in the absence of glucose, but no acetone or ethanol was produced under these conditions, reflecting the nature of the pathways involved in the production of butanol using only butyric acid. Ten grams per litre of butyric acid was found able to produce 13?g/L of butanol in the presence of 20?g/L of glucose, and 0.7?g/L butanol was produced in the absence of glucose. This study indicates the importance of the glucose to butyric acid ratio to the enhancement of butanol production.     

烟酸转磷酸核糖激酶和丙酮酸羧化酶共表达对大肠杆菌BA002产丁二酸的影响

大肠杆菌BA002是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。pncB是烟酸转磷酸核糖激酶 (NAPRTase) 的编码基因,通过过量表达pncB基因能够提高NAD(H)总量与维持合适的NADH/NAD+,从而恢复了厌氧条件下重组菌E. coli BA014 (BA002/pTrc99a-pncB) 的生长和产丁二酸的性能。然而,BA014在厌氧发酵过程中有大量丙酮酸积累,为进一步提高菌株的丁二酸生产能力,减少副产物丙酮酸的生成,共表达NAPRTase和来自于乳酸乳球菌 NZ9000中丙酮酸羧化酶 (PYC) 的编码基因pyc,构建了重组菌E. coli BA016 (BA002/pTrc99a-pncB-pyc)。3 L发酵罐结果表明,BA016发酵112 h后,共消耗了35.00 g/L的葡萄糖。发酵结束时,菌体OD600为4.64,产生了25.09 g/L丁二酸。通过共表达pncB和pyc基因,使BA016的丙酮酸积累进一步降低,丁二酸产量进一步提高。     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

共表达烟酸转磷酸核糖激酶和丙酮酸羧化酶对大肠杆菌NZN111产丁二酸的影响

构建了共表达烟酸转磷酸核糖激酶(NAPRTase)和丙酮酸羧化酶(PYC)的重组质粒pTrc99a-pncB-pyc,并考察了重组菌E.coli NZN111/pTrc99a-pncB-pyc生产丁二酸的能力。结果表明:重组菌NZN111/pTrc99a-pncB-pyc的NAPRTase和PYC的比酶活达到最高,分别为20.75和1.04 U/mg,同时,辅酶NADH、NAD+及NAD(H)总量达到最高。厌氧摇瓶发酵结果:48 h能够消耗17.5 g/L的葡萄糖生成14.08 g/L的丁二酸,而丙酮酸的产量大幅度降低,仅为0.11 g/L。本研究为基因工程菌大肠杆菌厌氧条件下发酵生产丁二酸提供了一定的基础。     

Accumulation of a cocoa-butter-like lipid by Yarrowia lipolytica cultivated on agro-industrial residues

Yarrowia lipolytica was cultivated on mixtures of saturated free fatty acids (an industrial derivative of animal fat called stearin), technical glycerol (the main by-product of bio-diesel production facilities), and glucose. The utilization of technical glycerol and stearin as co-substrates resulted in higher lipid synthesis and increased citric acid production than the combination of glucose and stearin. The lipids produced contained significant amounts of stearic acid (50-70%, wt/wt) and lower ones of palmitic (15-20%, wt/wt), oleic (7-20%, wt/wt), and linoleic (2-7%, wt/wt) acid. Single-cell oil having a composition similar to cocoa-butter up to 3.4 g/L was produced, whereas in some cases relatively increased citric acid quantities (up to 14 g/L) were excreted into the growth medium. The microorganism presented a high specificity for lauric, myristic, and palmitic acid, while a discrimination for the stearic acid was observed. As a conclusion, microbial metabolism could be directed by using mixtures of inexpensive saturated fats, glycerol, and glucose as co-substrates, in order to accumulate lipids with predetermined composition, e.g., cocoa-butter equivalents.     

Improving the production of S-adenosyl-L-methionine in Escherichia coli by overexpressing metk

S-adenosyl-L-methionine (SAM) has important applications in many fields including chemical therapy and pharmaceutical industry. In this study, the recombinant Escherichia coli strain was constructed for effective production of SAM by introducing the SAM synthase gene (metK). This strain produced 34.5?mg/L of SAM in basic medium in shake flask. Yeast extract, pH, and loaded volume had a significant positive effect on the yield of SAM. Their optimal values were 35?g/L, 7.5, and 30?mL, respectively. The final conditions optimized were as follows: glucose 20, g/L; peptone, 40?g/L; yeast extract, 35?g/L; NaCl, 10?g/L; MgSO₄, 1.2?g/L; L-methionine, 1?g/L; rotate speed, 220?rpm; loaded volume, 30?mL; inoculation, 1%; temperature, 37°C; and initial medium, pH 7.5. The recombinant strain produced 128.2?mg/L of SAM under the above conditions in shake flask. The production of SAM in a 5?L fermentor was also investigated. The maximal biomass of the recombinant strain was 60.4?g/L after the cells were cultured for 20?hr, and the highest yield of SAM was 300.9?mg/L after induction for 8?hr in a 5?L fermentor. This study provides a good foundation for the future production and use of SAM.     

Relieving effects of glycine and methionine from acetic acid inhibition in Escherichia coli fermentation

Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more beta-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h(-1), the methionine addition enhanced beta-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h (-1), the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. (c) 1993 John Wiley & Sons, Inc.     

Improvement of kojic acid production in Aspergillus oryzae B008 mutant strain and its uses in fermentation of concentrated corn stalk hydrolysate

A strain designated M866, producing kojic acid with a high yield, was obtained by combining induced mutation using ion beam implantation and ethyl methane sulfonate treatment of a wild type strain of Aspergillus oryzae B008. The amount of kojic acid produced by the strain M866 in a shaking flask was 40.2 g/L from 100 g/L of glucose, which was 1.7 times higher than that produced by wild strain (23.58 g/L). When the mixture of glucose and xylose was used as carbon source, the resulting kojic acid production was raised with the increasing of glucose ratios in the mixture. With concentrations of glucose at 75 g/L and xylose at 25 g/L mixed in the medium, the production of kojic acid reached 90.8 %, which was slightly lower than with glucose as the sole source of carbon. In addition, the kojic acid fermentation of the concentrated hydrolysate from corn stalk was also investigated in this study, the maximum concentration of kojic acid accumulated at the end of the fermentation was 33.1 g/L and this represents the yield based on reducing sugar consumed and the overall productivity of 0.36 g/g and 0.17 g/L/h, respectively.     

High cell density co-culture for production of recombinant hydrolases

Sugarcane bagasse is a residue with great potential as a feedstock for second-generation ethanol production. One of the approaches studied for making use of this material is the utilization of enzymes to hydrolyze the cell wall carbohydrates and generate fermentable sugars. These enzymes can be produced by cultivation of filamentous fungi or bacteria; however, the high production cost still represents a bottleneck to second-generation ethanol production. Expression of recombinant hydrolases through a co-culture strategy could be an interesting alternative for producing a recombinant cocktail at high levels of productivity that is tailor-made for each material to be hydrolyzed. In this study we evaluate the production of hydrolases by co-culturing two recombinant Escherichia coli, each expressing a specific hydrolase, β-1,3-1,4-glucanase or β-1,4-xylanase, both isolated from Bacillus subtilis. The cultures were conducted in bioreactors in batch and fed-batch mode in order to reach high cell densities. Co-culture in batch cultivation reached a dry cell weight of 10.4 g/L and volumetric activities of 31.96 U/mL and 11.89 U/mL for xylanase and endoglucanase, respectively. Fed-batch cultivation reached a dry cell weight of 60 g/L and the volumetric activities of xylanase and endoglucanase were respectively up to 5 and 1.3 times higher than those in batch mode. A competition assay indicates that no clone predominates over the other during cultivation. These results suggest that co-culture is a potential technique for producing recombinant hydrolase cocktails at lower cost than those associated with the production of a single culture.     

产琥珀酸重组大肠杆菌的发酵性能研究

研究了重组大肠杆菌JM001(△ppc)/pTrc99a-pck发酵产琥珀酸的性能,结果表明厌氧条件下其耗糖能力和产酸能力分别为对照菌株JM001的4.2倍和15.3倍。进一步优化发酵条件表明：采用接入菌泥的发酵方式比按照10%接种量转接厌氧发酵的效果要好,琥珀酸的对葡萄糖的质量收率提高了约10%,且副产物乙酸的量进一步降低。初始葡萄糖浓度高于60g/L时会对菌株的生长和产酸产生抑制,且浓度越高,抑制作用越明显。7L发酵罐放大实验中,整个厌氧发酵阶段葡萄糖的消耗速率为0.42g/(L.h),琥珀酸对葡萄糖的质量收率为67.75%,琥珀酸的生产强度为0.28g/(L.h)。     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate decarboxylase from Escherichia coli

Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis, GABA fermentation by C. glutamicum GAD cultured at 30°C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 ± 1.53 g/L extracellular GABA after 96 h. Addition of 0.1mM pyridoxal 5'-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1mM PLP, fermentation was performed in a flask at 30°C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 ± 0.88 g/L after 72 h with a space-time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source.     

Fermentative production of shikimic acid: a paradigm shift of production concept from plant route to microbial route

Different physiological and nutritional parameters affect the fermentative production of shikimic acid. In our study, Citrobacter freundii initially produced 0.62 g/L of shikimic acid in 72 h. However, when process optimization was employed, 5.11 g/L of shikimic acid was produced in the production medium consisting of glucose (5.0 %), asparagine (4.5 %), CaCO₃ (2.0 %), at pH 6.0, when inoculated with 6 % inoculum and incubated at 30 ± 1 °C, 200 rpm for 60 h. Preliminary fed-batch studies have resulted in the production of 9.11 g/L of shikimic acid on feeding the production medium by 20 g/L of glucose at 24 h of the fermentation run. Production of similar amount of shikimic acid was observed when the optimized conditions were employed in a 10-L bioreactor as obtained in shake flask conditions. A total of 9.11 g/L of shikimic acid was produced in 60 h. This is approximately 14.69-fold increase in shikimic acid production when compared to the initial un-optimized production conditions. This has also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.     

Continuous conversion of rice starch hydrolysate to 2-keto-D-gluconic acid by Arthrobacter globiformis C224

A continuous conversion process of rice starch hydrolysate to 2-keto-D-gluconic acid (2KGA) by Arthrobacter globiformis C224 was developed. Its feasibility for industrial application was also evaluated. Results showed that the initial cell concentration exceeding 1.25 g/L met the continuous 2KGA production at a stable dilution rate and media composition, while the dilution rate and feeding glucose concentration had a significant effect on 2KGA production performance. The optimal operating parameters were obtained as: 0.090/h of dilution rate and 171.0 g/L of feeding glucose concentration. Under these conditions, the steady state had a produced 2KGA concentration of 124.74 g/L, average volumetric productivity of 11.23 g/L/h, and yield of 0.97 g/g. In conclusion, continuous 2KGA production by the A. globiformis C224 strain would be a superior industrial process for the production of 2KGA in terms of its high 2KGA productivity and yield.     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading

Ethanol production by a recombinant bacterium from wheat straw (WS) at high solid loading by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid pretreated WS (150 g/L) after enzymatic saccharification was 86.3 ± 1.5 g/L. The pretreated WS was bio-abated by growing a fungal strain aerobically in the liquid portion for 16 h. The recombinant Escherichia coli strain FBR5 produced 41.1 ± 1.1 g ethanol/L from non-abated WS hydrolyzate (total sugars, 86.6 ± 0.3 g/L) in 168 h at pH 7.0 and 35 °C. The bacterium produced 41.8 ± 0.0 g ethanol/L in 120 h from the bioabated WS by SHF. It produced 41.6 ± 0.7 g ethanol/L in 120 h from bioabated WS by fed-batch SSF. This is the first report of the production of above 4% ethanol from a lignocellulosic hydrolyzate by the recombinant bacterium.