芽孢杆菌P38中乳酸脱氢酶对其产L-乳酸光学纯度的影响

【目的】研究芽孢杆菌(Bacillus sp.) P38中乳酸脱氢酶对其产高光学纯L-乳酸(光学纯度>99%)的影响。【方法】全基因组测序显示在该菌中存在3个乳酸代谢关键酶,分别为L-乳酸脱氢酶(L-LDH)、D-乳酸脱氢酶(D-LDH)和苹果酸或L-乳酸脱氢酶(M/L-LDH)。通过将这3个酶进行异源表达、纯化与酶学特性分析,结合Native-PAGE、实时荧光定量PCR等方法,初步确定该菌高产光学纯L-乳酸的机理。【结果】Bacillus sp. P38中L-LDH对丙酮酸的催化活性(Kcat/Km值)最高,分别是D-LDH的2.9倍和M/L-LDH的4.3倍。其中M/L-LDH主要起L-LDH的功能。Native-PAGE实验中未检测到D-LDH活性。Bacillus sp. P38所有发酵阶段ldhL的转录水平均高于ldhD和ldhM/L。【结论】L-LDH是Bacillus sp. P38产高光学纯L-乳酸的主要关键酶。     

Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-lactic acid

Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32. In order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L. helveticus CNRZ32 strains were constructed using gene replacement. One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species. The resulting strains were designated GRL86 and GRL89, respectively. In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter. The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type. The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain. Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology. The temperature and pH optima were 41 degrees C and pH 5.9. At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.     

Analysis of <Emphasis Type="Italic">ldh</Emphasis> genes in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> BL23: role on lactic acid production

Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid.     

L—乳酸脱氢酶基因克隆及功能分析

构建了一株产D,L-乳酸的乳杆菌(Lactobaeillus sp．)MD—1的基因库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJ144作为宿主,通过互补筛选分离克隆到乳酸脱氢酶基因(ldhL)。核酸序列分析表明,该基因以ATG为起始密码子编码316个氨基酸残基组成的蛋白质,预测的分子量为33．84kD;5′端存在典型的启动子结构,3′端的终止子是不依赖于ρ因子的转录终止子。ldhL编码的蛋白质有3个保守区域,其中Gly13～Asp50保守区域是NADH的结合位点,Asp73～Ile100和Asn123～Arg154保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低,核苷酸序列相似性最高仅为64．1％,氨基酸序列相似性最高仅为68．9％,是新的L—乳酸脱氢酶基因。     

Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis.

Diacetyl is an important food flavor compound produced by certain strains of citrate-metabolizing lactic acid bacteria. Citrate is converted to pyruvate, from which diacetyl is produced via intermediate alpha-acetolactate. This paper reports the cloning and analysis of the gene (aldB) encoding alpha-acetolactate decarboxylase from Lactococcus lactis MG1363. Deletion of the MG1363 chromosomal aldB gene was achieved by double crossover homologous recombination. The mutant strain was found to produce diacetyl; alpha-acetolactate decarboxylase activity was eliminated. Overexpression of the cloned ilvBN genes (encoding an alpha-acetolactate synthase) in the aldB deletion strain produced even higher levels of alpha-acetolactate, acetoin, and diacetyl.     

13C nuclear magnetic resonance analysis of glucose and citrate end products in an ldhL-ldhD double-knockout strain of Lactobacillus plantarum.

We have examined the metabolic consequences of knocking out the two ldh genes in Lactobacillus plantarum using 13C nuclear magnetic resonance. Unlike its wild-type isogenic progenitor, which produced lactate as the major metabolite under all conditions tested, ldh null strain TF103 mainly produced acetoin. A variety of secondary end products were also found, including organic acids (acetate, succinate, pyruvate, and lactate), ethanol, 2,3-butanediol, and mannitol.     

代谢工程改造野生耐酸酵母生产L-乳酸

以选育低pH条件下高产L-乳酸的酵母菌为目的,从自然样品中筛选分离得到一株能在pH 2.5 (乳酸调节) 的培养基中生长且不利用乳酸的酵母 (初步鉴定为木兰假丝酵母Candida magnolia);进一步将来源于米根霉As3.819的乳酸脱氢酶编码基因 (ldhA) 插入含有G418抗性基因的酵母穿梭载体,构建了重组质粒pYX212-kanMX-ldhA,电转化入野生型C. magnolia中,筛选获得了一株具有产L-乳酸能力的重组菌株C. magnolia-2;通过发酵实验表明,该重组菌产L-乳酸的最     

D-Lactate dehydrogenase gene (ldhD) inactivation and resulting metabolic effects in the Lactobacillus johnsonii strains La1 and N312.

Lactobacillus johnsonii La1, a probiotic bacterium with demonstrated health effects, grows in milk, where it ferments lactose to D- and L-lactate in a 60:40% ratio. The D-lactate dehydrogenase (D-LDH) gene (ldhD) of this strain was isolated, and an in vitro-truncated copy of that gene was used to inactivate the genomic copy in two strains, La1 and N312, by gene replacement. For that, an 8-bp deletion was generated within the cloned ldhD gene to inactivate its function. The plasmid containing the altered ldhD was transferred to L. johnsonii via conjugative comobilization with Lactococcus lactis carrying pAMbeta1. Crossover integrations of the plasmid at the genomic ldhD site were selected, and appropriate resolution of the cointegrate structures resulted in mutants that had lost the plasmid and in which the original ldhD was replaced by the truncated copy. These mutants completely lacked D-LDH activity. Nevertheless, the lower remaining L-LDH activity of the cells was sufficient to reroute most of the accumulating pyruvate to L-lactate. Only a marginal increase in production of the secondary end products acetaldehyde, diacetyl, and acetoin was observed. It can be concluded that in L. johnsonii D- and L-LDH are present in substantial excess for their role to eliminate pyruvate and regenerate NAD(+) and that accumulated pyruvate is therefore not easily redirected in high amounts to secondary metabolic routes.     

Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS(Mtl)). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Delta ldh Delta mtlA and Delta ldh Delta mtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo (13)C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS(Mtl). Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.     

Production of L-lactic acid by Rhizopus oryzae under oxygen limiting conditions

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.During aerobic growth, Rhizopus oryzae produces L-lactic acid from lactate dehydrogenase mediated reduction of pyruvate, while O2 limiting conditions yield primarily ethanol. A mutant was isolated that expressed only 5% of the wild type alcohol dehydrogenase activity under O2 limiting conditions and produced nearly 40 g lactic acid/l in 70 h. This is almost a ten-fold increase in lactic acid production when compared to the parent strain.     

Disruption of lactate dehydrogenase through homologous recombination to improve bioethanol production in Thermoanaerobacterium aotearoense

To enhance ethanol production in Thermoanaerobacterium aotearoense, the lactate dehydrogenase (ldh) gene, which is responsible for lactic acid production in a key branch pathway, was successfully disrupted via homologous recombination. ldh-up and ldh-down were designed and amplified based on JW/SL-YS485-AY 278026, and they were subsequently used as homologous fragments with an inserted erythromycin resistance gene to construct the targeted vector based on pBLUESCRIPT II SK(+). Southern hybridization and PCR-based assay definitely confirmed that the ldh gene in the Δldh mutant was disrupted by the insertion of the erythromycin resistance gene. Compared with the wild type, the Δldh mutant exhibited increases of 31.0% and 31.4% in cell yield under glucose and xylose cultivation, respectively, probably because knocking out the ldh gene results in increased acetate and ATP levels. Knockout of lactate dehydrogenase produced 2.37- and 2.1-fold increases in the yield of ethanol (mole/mole substrate) under glucose and xylose cultivation, respectively. Moreover, no lactic acid was detected in Δldh mutant fermentation mixtures (detection limit of HPLC: 0.5 mM), but lactic acid was readily detected for growth of the wild-type strain on both glucose and xylose, with final concentrations up to 59.24 mM and 56.06 mM, respectively. The success of this process thoroughly demonstrates the methodological possibility of gene knockout through homologous recombination in Thermoanaerobacterium.     

利用代谢工程构建D-甘露醇生产菌株

D-甘露醇广泛应用于食品、制药、化学品工业等领域。从野生型大肠杆菌出发,将来自假肠膜明串珠菌Leuconostoc pseudomesenteroides ATCC 12291菌株的甘露醇脱氢酶与果糖转运蛋白编码基因整合到大肠杆菌ATCC 8739的染色体中,并失活其他的发酵途径 (丙酮酸甲酸裂解酶、乳酸脱氢酶、富马酸还原酶、乙醇脱氢酶、甲基乙二醛合成酶和丙酮酸氧化酶) ,构建了一株遗传稳定的D-甘露醇生产菌株。使用无机盐培养基和葡萄糖果糖作为混合碳源,厌氧发酵6 d,D-甘露醇产量达1.2 mmol/L。基于细胞生长和D-甘露醇合成的偶联,进一步通过代谢进化技术提高细胞合成D-甘露醇的生产能力。经过80代的驯化,D-甘露醇产量提高了2.6倍,甘露醇脱氢酶的活性提高了2.8倍。构建获得的遗传稳定的工程菌能直接发酵糖生产D-甘露醇,不需添加抗生素、诱导剂和甲酸,在工业化生产时有一定优势。     

米根霉乙醇脱氢酶突变株的筛选及其锌镁离子的调控研究

利用亚硝基胍(NTG)对米根霉As3.3461进行诱变,在含丙烯醇0.6%的YPD平板上筛选获得21株乙醇含量降低的突变株,其中突变株HBF-12乳酸产量最高。与出发菌株相比,突变株HBF-12的乙醇产量和乙醇脱氢酶(ADH)活力分别降低了73.6%和76%,乳酸产量和乳酸脱氢酶(LDH)活力分别提高了41.2%和19.6%。研究Zn2 与Mg2 对HBF-12中ADH与LDH活性的调控,结果显示Zn2 对ADH有强烈的激活作用,但抑制LDH活性;Mg2 则轻微抑制ADH活性,促使LDH活性增强。考察两种离子影响末端产物乙醇与乳酸形成的实验说明:培养基中Zn2 浓度与乳酸积累基本上呈负相关性,与乙醇积累呈正相关性,浓度降低有利于生物量积累;Mg2 浓度增加可以促进乳酸积累和生物量增加,对于乙醇积累无明显作用。发酵培养基中添加0.01%Zn2 、0.04%Mg2 ,突变株产酸可达96.21g/L。     

d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici

The microbial production of L-(+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of L-(+) and D-(-) isomers. For most uses of PLA, the L-(+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions (focA-pflB frdBC adhE ackA ldhA). This strain was constructed from a D-(-)-lactic acid-producing strain, SZ63 (focA-pflB frdBC adhE ackA), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an L-lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in L-lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native D-lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased L-lactate dehydrogenase activity. SZ85 produced L-lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for L-lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes.     

谷氨酸棒杆菌1dh基因的敲除

谷氨酸棒杆菌中ldh基因编码乳酸脱氢酶,可催化丙酮酸转化生成乳酸.利用重叠延伸PCR的方法,获得中间缺失部分序列的dldh基因片段,将其与载体pk 18mobsacB连接,转化大肠杆菌感受态,筛选出阳性转化子后,转化谷氨酸棒杆菌ATCC 13032感受态细胞.分别在卡那霉素抗性平板及10％蔗糖平板上进行两次筛选,利用PCR方法鉴定,成功获得ldh基因缺失的谷氨酸棒杆菌突变株ATCC 13032-(4)ldh.应用荧光定量PCR检测,ATCC 13032-(z)ldh中的ldh基因在转录水平与野生型菌株ATCC 13032相比,相对表达量为O.ldh基因的敲除对菌株的生长造成了一定的影响.