Environmental conditions during glycerol bioconversion affect 3-hydroxypropionic acid bioproduction by Limosilactobacillus reuteri DSM 17938

3-Hydroxypropionic acid (3-HP) is a platform molecule whose biological production was carried out by the bacterium Limosilactobacillus reuteri according to a two-step process: first, a growth phase in batch mode on glucose, then a glycerol bioconversion into 3-HP in fed-batch mode. With the objective of improving 3-HP bioproduction, this study aimed at defining the operating conditions during the bioconversion phase that increases the bioproduction performance. A central composite rotatable design allowed testing various pH levels and specific glycerol feeding rates. By establishing response surfaces, optimal conditions have been identified that were different depending on the considered output variable (final 3-HP quantity, 3-HP production yield and production rate). Of them, 3-HP final quantity and 3-HP production yield were maximized at pH 6.0 and at specific glycerol feeding rates of 60 and 55 mg_gly g_CDW⁻¹ h⁻¹, respectively. The specific 3-HP production rate was the highest at the upper limit of the specific substrate feeding rate (80 mg_gly g_CDW⁻¹ h⁻¹) but was not affected by the pH. An additional experiment was carried out at pH 6.0 and a specific glycerol feeding rate of 80 mg_gly g_CDW⁻¹ h⁻¹ to validate the previous observations. In conclusion, the results showed a significant improvement of 3-HP concentration by 13%, of specific production rate by 34% and of 3-HP volumetric productivity by 39%, as compared to the initial values.     

Identification and characterization of the propanediol utilization protein PduP of Lactobacillus reuteri for 3-hydroxypropionic acid production from glycerol

Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD⁺ and NADP⁺ as a cofactor. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37 °C. The K _m and V _max values for propionaldehyde in the presence of NAD⁺ were 1.18 mM and 0.35 U mg⁻¹, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild-type strain (from 0.18 g L⁻¹ to 0.72 g L⁻¹) under shake-flask culture conditions, and the highest titer (1.38 g L⁻¹ 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.     

Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.     

Effect of glucose on glycerol bioconversion by Lactobacillus reuteri

The impact of glucose on glycerol metabolism, especially on 3-hydroxypropionaldehyde (3-HPA) accumulation by resting cells of Lactobacillus reuteri has been investigated. Two systems were used in the study: MRS(-) (modified MRS - omitting glucose, acetate and Tween 80) and distilled water (H(2)O). In MRS(-), addition of glucose enhanced glycerol metabolism in resting cells of L. reuteri, consequently increasing the accumulation of 3-HPA by regulating the NAD/NADH ratio. Enhanced glycerol metabolism correlated positively with the concentration of glucose. NADH produced during glucose metabolism was preferentially reoxidized to NAD by the reduction of 3-HPA to 1,3-propanediol; an adequate supply of glycerol therefore outweighed the repression of glucose on the accumulation of 3-HPA. At a molar ratio of glucose to glycerol no greater than 0.33, accumulation of 3-HPA was favored. In non-growing medium (H(2)O), addition of glucose seemed to be counter-productive with respect to 3-HPA accumulation. Lactate had a positive impact on glycerol metabolism, presumably by altering the redox flux, resulting in enhanced 3-HPA accumulation in both MRS(-) and H(2)O systems.     

Production and stability of 3-hydroxypropionaldehyde in Lactobacillus reuteri

3-Hydroxypropionaldehyde (3-HPA) is considered as a potent antimicrobial substance. Exploration of its application as a food preservative or as a therapeutic auxiliary agent has been documented in the literature. In the present work, factors that may impact on 3-HPA accumulation by Lactobacillus reuteri and on the stability of 3-HPA were investigated. Three media - H(2)O, milk and MRS broth - were chosen as test systems. Data indicated that 3-HPA accumulation in resting cells of L. reuteri in a two-step fermentation is greatly affected by temperature, pH, cell age and biomass as well as components in the test system. Within 2 h of incubation, 170 mM 3-HPA could be produced with a cell dry weight of 30 g/l, representing 85% of the glycerol supplied (200 mM) in H(2)O. The presence of glycerol during cell growth increased the productivity of 3-HPA by resting cells. In general, 3-HPA is much more stable in H(2)O than in milk and MRS. Factors that enhanced accumulation of 3-HPA did not simply show the same positive impact on the stability of 3-HPA. Thus, for defined applications, factors affecting production and stability of 3-HPA should be evaluated separately.     

An integrated process for the production of 1,3-propanediol,lactate and 3-hydroxypropionic acid by an engineered Lactobacillus reuteri

The process economy of food grade 1,3-propanediol (1,3-PD) production by GRAS organisms like Lactobacillus reuteri (L. reuteri), is negatively impacted by the low yield and use of expensive feedstocks. In order to improve the process economy, we have developed a multiproduct process involving the production of three commercially important chemicals, namely, 1,3-PD, lactate and 3-Hydroxypropionic acid (3-HP), by engineered L. reuteri. The maximum 1,3-PD and lactate titer of 41 g/L and 31 g/L, with a volumetric productivity of 1.69 g/L/h and 0.67 g/L/h were achieved, respectively. The maximum 3-HP titer of 5.2 g/L with a volumetric productivity of 1.3 g/L/h, was obtained by biotransformation using cells recovered from the repeated fed-batch process. The volumetric productivity of 1,3-PD obtained in this study is the highest ever reported for this organism. Further cost reduction can be achieved by using waste feedstocks like milk whey, biomass hydrolysate, and crude glycerol.     

Production of high amounts of 3-hydroxypropionaldehyde from glycerol by Lactobacillus reuteri with strongly increased biocatalyst lifetime and productivity

3-hydroxypropionaldehyde (3HPA) is a promising versatile substance derived from the renewable feedstock glycerol. It is a product of glycerol metabolism in Lactobacillus reuteri. Because of toxic effects, the biotechnological production is poor. In this work the biocatalyst lifetime and product formation could be drastically increased. In the established two-step process already applied, cells are grown in the first step under anaerobic conditions, and in the second step the immobilised or suspended biocatalyst is used for 3HPA-production under strict anaerobic conditions. In the first step it was possible to reach a biomass concentration of 5.5g CDW/L (OD(600)≈23.4). In the second step, normally, 3HPA accumulates to a toxic concentration and the reaction stops in less than 60min because of the interaction of 3HPA with cell components. To prevent this, the toxic product is bound to the newly found scavenger carbohydrazide to form the hydrazone. For the first time it was possible to recycle the immobilised biocatalyst for at least ten cycles (overall life time>33hours) in a repeated batch biotransformation with an overall production of 67g 3HPA. The optimal pH-value was between 6.8 and 7.2 at an optimal temperature of 40-45°C. In a single batch biotransformation with suspended resting cells it was possible to produce 150g/L 3HPA as carbohydrazone at an overall productivity of 10.7gL(-1)hours(-1). In a single fed-batch biotransformation at 45°C 138g/L glycerol was converted into 108g/L 3HPA with an overall productivity of 21.6gL(-1)hours(-1). This is the highest 3HPA concentration and productivities reported so far for the microbial production of 3HPA from glycerol.     

Production of 3-hydroxypropionic acid from glycerol by acid tolerant Escherichia coli

The biological production of 3-hydroxypropionic acid (3-HP) has attracted significant attention because of its industrial importance. The low titer, yield and productivity, all of which are related directly or indirectly to the toxicity of 3-HP, have limited the commercial production of 3-HP. The aim of this study was to identify and select a 3-HP tolerant Escherichia coli strain among nine strains reported to produce various organic acids efficiently at high titer. When transformed with heterologous glycerol dehydratase, reactivase and aldehyde dehydrogenase, all nine E. coli strains produced 3-HP from glycerol but the level of 3-HP production, protein expression and activities of the important enzymes differed significantly according to the strain. Two E. coli strains, W3110 and W, showed higher levels of growth than the others in the presence of 25 g/L 3-HP. In the glycerol fed-batch bioreactor experiments, the recombinant E. coli W produced a high level of 3-HP at 460 ± 10 mM (41.5 ± 1.1 g/L) in 48 h with a yield of 31 % and a productivity of 0.86 ± 0.05 g/L h. In contrast, the recombinant E. coli W3110 produced only 180 ± 8.5 mM 3-HP (15.3 ± 0.8 g/L) in 48 h with a yield and productivity of 26 % and 0.36 ± 0.02 g/L h, respectively. This shows that the tolerance to and the production of 3-HP differ significantly among the well-known, similar strains of E. coli. The titer and productivity obtained with E. coli W were the highest reported thus far for the biological production of 3-HP from glycerol by E. coli.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

A novel cell modification method used in biotransformation of glycerol to 3-HPA by Lactobacillus reuteri

The aim of the present study was to develop a new cell modification method to facilitate the cell separation from broth. In order to reduce the transfer limitation of substrate and product caused by general immobilization methods in the following biotransformation of glycerol, the carboxyl-functioned superparamagnetic nanoparticle (MNP) was directly attached to the surface of Lactobacillus reuteri for 3-hydroxypropionealdehyde producing. The modification process could be finished in several minutes by just adding MNP fluid into the bulk fermentation broth. The modified cells could be rapidly separated from the solution with the aid of magnetic field. The interaction between cell and MNP was shown by electron microscopy. The efficiency of the cells attached by MNPs for transformation of various concentrations of glycerol (100–400 mM) was studied at various temperatures (25–40 °C) and pH levels (5.8–7.5) with different cell concentrations (7.5–30 g/L). The 3- hydroxypropionealdehyde (HPA)/glycerol molar conversion under optimal condition (30 °C and pH 7) reached 70 %. The inactive modified cell could be reactivated easily by fresh medium and recovered the ability of glycerol conversion. MNPS distributing on cell surface had little adverse effect on cell activity. The modification method simplified the two-step production of 3-HPA by resting L. reuteri. The method of MNPs attached to cell surface is totally different from the traditional immobilization method in which the cell is attached to or entrapped in big carrier. The results obtained in this study showed that carboxyl-functioned MNP could be directly used as cell modification particle and realized cell recycle with the aid of magnetic field in bioprocess.     

利用甘油发酵耦联生产3-羟基丙酸及1,3-丙二醇重组菌的构建及筛选

在肺炎克雷伯杆菌(Klebsiella pneumoniae)代谢甘油生产1,3-丙二醇(1,3-PD)的过程中,为了减少有毒中间产物3-羟基丙醛(3-HPA)的积累,可将其转化为3-羟基丙酸(3-HP),从而实现1,3-丙二醇和3-羟基丙酸的联产。克隆来自于酿酒酵母的NAD+依赖型的乙醛脱氢酶(ALDH)的基因aldh4,构建了表达载体pKP-aldh,转化K.pneumoniae,得到了有效表达乙醛脱氢酶的重组肺炎克雷伯杆菌(K.pneumoniae A+)。在此基础上,使用紫外诱变联合菌种驯化的方法对K.pneumoniae A+进行筛选,获得了可耐受较高3-HP浓度(≥35 g/L)的重组肺炎克雷伯杆菌K.pneumoniae A+5-3。发酵实验结果表明,K.pneumoniae A+5-3可将3-HPA转化为3-HP,能够同时利用甘油耦联生产3-HP和1,3-PD,产量分别达到5.0 g/L和74.5 g/L。     

氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸

3-羟基丙酸是一种潜在的重要化工产品,可作为中间体合成多种有经济价值的工业用化合物。文中利用氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸。首先在50 mL摇瓶中(转化体系为10 mL)考察细胞加入量、底物和产物浓度等对催化反应的影响。在此基础上,在2 L鼓泡塔中(转化体系为1 L),采取适当的补料方式和生物转化与分离相耦合的手段解除抑制,以提高目标产物终浓度。结果表明:高底物和产物浓度通过降低反应初速度抑制转化的进行,并确定了最佳催化反应条件为6 g/L菌体量,pH 5.5。利用流加补料方式维持反应体系中底物浓度在15~20 g/L,经过60 h的反应,3-羟基丙酸的浓度达到60.8 g/L,生产强度为1.0g/(L.h),转化率为84.3%。采用生物转化与分离相耦合的方法,经过50 h的转化反应,3-羟基丙酸的总产量达76.3 g/L,生产强度为1.5 g/(L.h),转化率83.7%。研究结果对利用氧化葡萄糖酸杆菌的不完全氧化醇类化合物特性实现其在工业生物催化中的应用具有一定的指导意义。     

罗伊乳酸杆菌甘油脱水酶基因的克隆及其在大肠杆菌中的表达

迅速升温的生物柴油投资热导致了其副产物甘油的大量积累,这一现状使得开发和利用甘油生产各种精细化工产品备受关注。本实验通过构建基因工程菌来生物转化甘油生产3-羟基丙醛,为甘油下游产品的开发开辟了一条新途径。3-羟基丙醛是一种重要的化学中间体,同时也是一种有效的抗菌剂和生物组织的固定剂,在化学工业中具有广泛的应用前景。实验主要利用甘油脱水酶N末端序列,并根据NCBI中公布的甘油脱水酶的氨基酸序列设计了一对克隆引物,并以菌株罗伊乳酸杆菌Lactobacillus reuteri的基因组DNA为模板进行PCR扩增,获得约为1.6kb的片段,将其克隆到T载体上进行测序,对测序结果进行分析,重新设计两端含有EcoRI和HindIII酶切位点的表达引物,利用PCR扩增得到了甘油脱水酶基因,该基因片段长度为1674bp,编码558个氨基酸。将所得片段定向克隆到pET28b载体中,并转化至大肠杆菌BL21感受态细胞中。经IPTG诱导后,进行SDS-PAGE电泳,在约65kD处检测出一蛋白表达条带,另外还对该重组菌进行比活力测定,最高比活力可达1.14U/mg,比野生型菌株提高了86.88%。     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Aldehyde dehydrogenase activity is important to the production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae

3-Hydroxypropionic acid (3-HP) can be produced from glycerol via two enzymatic reactions catalyzed by a coenzyme B₁₂-dependent glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) in Klebsiella pneumoniae. As the intracellular GDHt activity in K. pneumoniae is high, the overall rate of 3-HP production is controlled by the ALDH activity. To examine the effect of different ALDH activity on 3-HP production, three different ALDHs, AldH from Escherichia coli (EaldH), PuuC from K. pneumoniae (PuuC) and KGSADH from Azospirillum brasilense (KGSADH), were overexpressed and compared in various recombinant K. pneumoniae strains. In addition, the genes encoding DhaT and YqhD, which are responsible for the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PDO), were disrupted individually from K. pneumoniae to enhance the carbon flux from 3-HPA to 3-HP. When the ALDH activity was measured in various recombinant K. pneumoniae, KGSADH showed the highest crude cell activity of 8.0 U/mg protein, which was 2 and 4 times higher than that of PuuC and EaldH, respectively. The different ALDH activities had a significant effect on 3-HP production in a flask culture containing 100 mM glycerol, and K. pneumoniae ΔdhaT (KGSADH) resulted in the highest titer (64 mM) among the nine recombinant strains (three ALDH × three host strains; one wild type and two mutants). In glycerol fed-batch bioreactor cultivation, K. pneumoniae ΔdhaT (KGSADH) exhibited 3-HP production at >16 g/L in 48 h with a glycerol carbon yield of >40%. In comparison, K. pneumoniae ΔdhaT (PuuC) produced only 11 g/L 3-HP in 48 h with a yield of >23%. This study demonstrates that a high ALDH activity is essential for the effective production of 3-HP from glycerol with recombinant K. pneumoniae.     

透性化嗜酸乳杆菌细胞转化亚油酸为共轭亚油酸的反应动力学

本实验旨在研究透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的反应动力学。探讨了细胞浓度、底物浓度、反应体系pH值和温度等因素对生物转化共轭亚油酸反应速度的影响;建立了透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的动力学模型。结果表明,透性化嗜酸乳杆菌细胞有利于共轭亚油酸的生物转化,最适细胞浓度、pH值和反应温度分别为10×1010ufc/mL、4.5和45℃;生物转化共轭亚油酸存在底物抑制现象,当亚油酸的浓度为0.6mg/mL时,反应速度达到最大值17.8μg/(mL·min)。在低亚油酸浓度下,反应初始阶段的反应规律与经典米氏方程相符,而在高亚油酸浓度下,存在底物抑制现象。在最适反应条件下建立了动力学模型,模型基本反映了共轭亚油酸的生物转化特性。     

Co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase

The co-production of 3-hydroxypropionic acid (3HP) and 1,3-propanediol (PDO) from glycerol was studied using the resting cells of a recombinant Klebsiella pneumoniae J2B strain that overexpresses an aldehyde dehydrogenase (KGSADH). Active biomass was produced in a mineral salt medium containing yeast extract and glycerol under a range of aeration conditions, and shifted to potassium phosphate buffer containing glycerol for bioconversion. The microaerobic or anaerobic conditions were favorable for both the production of active biomass and subsequent bioconversion. At the flask level, the recombinant strain (2.0?g?CDW/L) grown under microaerobic conditions produced 43.2?mM 3HP and 59.0?mM PDO from glycerol (117?mM) in 30?min with a cumulative yield of 0.87?(mol/mol). The fed-batch bioconversion, which was performed in a 1.5-L bioreactor with 1.0?g?CDW/L at a constant pH?7.0 under anaerobic conditions, resulted in 125.6?mM 3HP and 209.5?mM PDO in 12?h with a cumulative overall productivity, yield, and maximum specific production rate of 27.9?mmol/L/h, 0.71 (mol/mol), and 128.5?mmol/g CDW/h, respectively. Lactate, succinate and 2,3-butanediol were the major by-products, whereas the production of acetate and ethanol was marginal. This is the first report of the simultaneous production of 3HP and PDO from glycerol using a resting cell system.