以甘油为底物利用重组大肠杆菌生产聚3-羟基丙酸

【目的】解决前期研究中所构建的以甘油为底物合成聚3-羟基丙酸(P3HP)的代谢途径中存在两个主要的问题——细胞内还原力不平衡和质粒丢失,以提高P3HP的产量。【方法】克隆来源于肺炎克雷伯氏菌的1,3-丙二醇(1,3-PDO)氧化还原酶基因,构建P3HP和1,3-PDO联产的菌株,解决细胞内还原力不平衡的问题。利用自杀性载体系统介导的同源重组技术,将甘油脱水酶及其激活因子的基因整合到大肠杆菌基因组中,提高质粒的稳定性。同时,对发酵条件进行优化。【结果】菌种改造和发酵条件优化显著提高了P3HP产量,在摇瓶条件下到达2.7 g/L,比以前的报道提高2倍,并可同时得到2.4 g/L 1,3-PDO。【结论】该重组大肠杆菌合成P3HP的产量得到提高,具有较好的工业化生产前景。     

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L^-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.     

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.     

Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis

3-Hydroxypropionate (3HP) is an important platform chemical, and four 3HP biosynthetic routes were reported, in which the malonyl-CoA pathway has some expected advantages but presented the lowest 3HP yield. Here, we demonstrated that this low yield was caused by a serious functional imbalance between MCR-C and MCR-N proteins, responsible for the two-step reduction of malonyl-CoA to 3HP. Then we minimized the enzyme activity imbalance by directed evolution of rate-limiting enzyme MCR-C and fine tuning of MCR-N expression level. Combined with culture conditions optimization, our engineering approaches increased the 3HP titer 270-fold, from 0.15 g/L to 40.6 g/L, representing the highest 3HP production via malonyl-CoA pathway so far. This study not only significantly improved the 3HP productivity of recombinant Escherichia coli strain, but also proved the importance of metabolic balance in a multistep biosynthetic pathway, which should be always considered in any metabolic engineering study.     

甲羟戊酸途径限速步骤研究及其在产番茄红素重组大肠杆菌中的应用

甲羟戊酸途径(MVA途径)被引入重组大肠杆菌中,能够提高重组大肠杆菌中萜类化合物的合成能力。但因重组大肠杆菌中萜类化合物合成途径中间产物积累,导致细胞生长和萜类化合物合成受到限制。本研究在稳定表达MVA途径以及优化2-甲基-D-赤藻糖醇-4-磷酸途径(MEP途径)、番茄红素合成途径关键基因表达的重组大肠杆菌LYC103中,用质粒高表达MVA途径和番茄红素合成途径关键基因,挖掘该途径的限速步骤。结果表明,ispA、crtE、mvaK1、idi和mvaD基因过表达后,细胞生长没有明显变化,番茄红素产量依次提高了13.5%、16.5%、17.95%、33.7%和61.1%,说明这几个基因可能是合成番茄红素的限速步骤。mvaK1、mvaK2、mvaD三个基因在同一操纵子上,用mRNA稳定区(RNA stabilizing region)进行启动子文库(mRSL)调控mvaK1,相当于对3个基因同时调控。用高效基因组编辑技术(CAGO)对mvaK1基因的mRNA稳定区进行启动子文库的调控,得到菌株LYC104。番茄红素产量与对照菌株LYC103相比增加了2倍,细胞生长提高了32%。然后,利用CRISPR-Cas9技术在染色体lacZ位点整合idi基因,得到LYC105菌株。与出发菌株LYC103相比,细胞生长提高了147%,番茄红素产量增加了2.28倍。本研究在染色体上具有完整MVA途径的基础上,利用质粒高表达单个基因挖掘限速步骤,用同源重组方法整合限速基因、解除限速,为代谢工程构建高产菌株提供新策略。     

Assessment of Synthesis Machinery of Two Antimicrobial Peptides from Paenibacillus alvei NP75

Paenibacillus alvei NP75, a Gram-positive bacterium, produces two different antimicrobial peptides, paenibacillin N and P, which has potent antimicrobial activity against many clinical pathogens. The synthesis pattern of these antimicrobial peptides by P. alvei NP75 was studied extensively. The results were outstanding in a way that the paenibacillin N was synthesized irrespective of the growth of bacteria (non-ribosomal mediated), whereas paenibacillin P production was carried out by ribosomal mediated. In addition to the antimicrobial peptides, P. alvei NP75 also produces an immunogenic extracellular protease to defend itself from its own antimicrobial peptide, paenibacillin P. Furthermore, this immunogenic protease production was impaired by the addition of protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). The sodium dodecyl sulfate (SDS) treated strain (mutant) failed to produce paenibacillin P, whereas the production of neither paenibacillin N nor the protease was affected by the plasmid curing. The plasmid curing studies that divulge the genes responsible for the synthesis of paenibacillin N and protease were found to be genome encoded, and paenibacillin P was plasmid encoded. We are reporting, first of its kind, the co-production of two different antimicrobial peptides from P. alvei NP75 through non-ribosomal and ribosomal pathways that could be used as effective antibiotics.

     

Biosynthetic pathway for poly(3-Hydroxypropionate) in recombinant Escherichia coli

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.     

Evaluation of 3-hydroxypropionate biosynthesis in vitro by partial introduction of the 3-hydroxypropionate/4-hydroxybutyrate cycle from <Emphasis Type="Italic">Metallosphaera sedula</Emphasis>

The chemical 3-hydroxypropionate (3HP) is an important starting reagent for the commercial synthesis of specialty chemicals. In this study, a part of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula was utilized for 3HP production. To study the basic biochemistry of this pathway, an in vitro-reconstituted system was established using acetyl-CoA as the substrate for the kinetic analysis of this system. The results indicated that 3HP formation was sensitive to acetyl-CoA carboxylase and malonyl-CoA reductase, but not malonate semialdehyde reductase. Also, the competition between 3HP formation and fatty acid production was analyzed both in vitro and in vivo. This study has highlighted how metabolic flux is controlled by different catalytic components. We believe that this reconstituted system would be valuable for understanding 3HP biosynthesis pathway and for future engineering studies to enhance 3HP production.     

Expression of NADH-oxidases enhances ethylene productivity in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bacterial EFE

Ethylene is a major petrochemical for which biotechnological production methods are an attractive alternative. Here we use a system based on a bacterial ethylene forming enzyme (EFE) expressed in Saccharomyces cerevisiae. Metabolic modelling performed in a previous study identified re-oxidation of NADH as a factor limiting ethylene production in S. cerevisiae. In line with this, we here found that strains with multicopy plasmid expression of the heterologous oxidases nox and Aox1 led to significantly increased specific ethylene productivity, up 12 and 36%, respectively, compared to the control strain with empty plasmid. However the productivity and yield was only improved in the AOX expressing strain compared to that of the control strain. Both oxidase expressing strains also exhibited increased respiration rates compared to the reference strain, with specific oxygen consumption rates being roughly doubled in both strains. The AOX strain furthermore exhibited a significant increase in the EFE substrate 2-oxoglutarate formation compared to the reference strain, linking an improvement in ethylene production to both increased respiratory capacity and increased substrate availability, thereby corroborating our previous finding.     

Complete Genome Sequence of Enterococcus mundtii QU 25, an Efficient l-(+)-Lactic Acid-Producing Bacterium

Enterococcus mundtii QU 25, a non-dairy bacterial strain of ovine faecal origin, can ferment both cellobiose and xylose to produce l-lactic acid. The use of this strain is highly desirable for economical l-lactate production from renewable biomass substrates. Genome sequence determination is necessary for the genetic improvement of this strain. We report the complete genome sequence of strain QU 25, primarily determined using Pacific Biosciences sequencing technology. The E. mundtii QU 25 genome comprises a 3 022 186-bp single circular chromosome (GC content, 38.6%) and five circular plasmids: pQY182, pQY082, pQY039, pQY024, and pQY003. In all, 2900 protein-coding sequences, 63 tRNA genes, and 6 rRNA operons were predicted in the QU 25 chromosome. Plasmid pQY024 harbours genes for mundticin production. We found that strain QU 25 produces a bacteriocin, suggesting that mundticin-encoded genes on plasmid pQY024 were functional. For lactic acid fermentation, two gene clusters were identified—one involved in the initial metabolism of xylose and uptake of pentose and the second containing genes for the pentose phosphate pathway and uptake of related sugars. This is the first complete genome sequence of an E. mundtii strain. The data provide insights into lactate production in this bacterium and its evolution among enterococci.     

Sequencing and analysis of plasmids pAV1 and pAV2 ofAcinetobacter venetianus VE-C3 involved in diesel fuel degradation

Acinetobacter venetianus strain VE-C3 was isolated in the Venice lagoon (Italy) as a strain able to degrade diesel fuel oil. This strain possesses genes of the alkane monoxygenase complex responsible forn-alkane degradation and carries two plasmids, pAV1 (10820 bp) and pAV2 (15135 bp), which were supposed from the analysis of Alk mutant strains to harbour genetic determinants for hydrocarbon degradation. In this work we determined the nucleotide sequence of both plasmids and showed the presence of a putative aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on plasmid pAV2, and of an ORF similar toalkL gene present on pAV1 plasmid. These data, combined with genetic reports indicating that strains lacking one of the two plasmids or carrying transposon insertion on pAV1, are defective inn-alkane degradation, suggest a complex genomic organisation of genes involved in alkane degradation inA. venetianus VE-C3. In this bacterium these genes are carried by both the chromosome and the plasmids, while inAcinetobacter sp. strain ADP1 and M1 all the genes for alkane monoxygenase complex are located only on the chromosome.     

Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts

Two volatile thiols, 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical varietal aroma compounds in Sauvignon Blanc (SB) wines. These aromatic thiols are not present in the grape juice but are synthesized and released by the yeast during alcoholic fermentation. Single deletion mutants of 67 candidate genes in a laboratory strain of Saccharomyces cerevisiae were screened using gas chromatography mass spectrometry for their thiol production after fermentation of SB grape juice. None of the deletions abolished production of the two volatile thiols. However, deletion of 17 genes caused increases or decreases in production by as much as twofold. These 17 genes, mostly related to sulfur and nitrogen metabolism in yeast, may act by altering the regulation of the pathway(s) of thiol production or altering substrate supply. Deleting subsets of these genes in a wine yeast strain gave similar results to the laboratory strain for sulfur pathway genes but showed strain differences for genes involved in nitrogen metabolism. The addition of two nitrogen sources, urea and di-ammonium phosphate, as well as two sulfur compounds, cysteine and S-ethyl-L-cysteine, increased 3MH and 3MHA concentrations in the final wines. Collectively these results suggest that sulfur and nitrogen metabolism are important in regulating the synthesis of 3MH and 3MHA during yeast fermentation of grape juice.     

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.     

Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters

The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda⁻) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed.     

Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production

Limonene is a valuable monoterpene used in the production of several commodity chemicals and medicinal compounds. Among them, perillyl alcohol (POH) is a promising anti-cancer agent that can be produced by hydroxylation of limonene. We engineered E. coli with a heterologous mevalonate pathway and limonene synthase for production of limonene followed by coupling with a cytochrome P450, which specifically hydroxylates limonene to produce POH. A strain containing all mevalonate pathway genes in a single plasmid produced limonene at titers over 400 mg/L from glucose, substantially higher than has been achieved in the past. Incorporation of a cytochrome P450 to hydroxylate limonene yielded approximately 100 mg/L of POH. Further metabolic engineering of the pathway and in situ product recovery using anion exchange resins would make this engineered E. coli a potential production platform for any valuable limonene derivative.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Enhanced production of aureofuscin by over-expression of AURJ3M, positive regulator of aureofuscin biosynthesis in Streptomyces aureofuscus

Aims: The production of aureofuscin is very low in the wild‐type strain. We attempt to increase the production of aureofuscin by over‐expression of a controlling gene in the wild‐type strain. Methods and Results: The aurj3M gene was PCR‐amplified from Streptomyces aureofuscus SYAU0709, ligated into vector pMD19 and sequenced. The predicted translation of the 579‐bp cloned fragment was 97% similar to pimM from Streptomyces natalensis, which has an N‐terminal PAS domain and a LuxR‐type C‐terminal helix–turn–helix. Recombinant bacterial strains were constructed by transforming SYAU0709 with an expression plasmid (pBJJ3M) that contained aurj3M, thereby increasing the number of aurj3M gene copies. Conclusions: Bioassays for the antibiotic compound aureofuscin indicated that the recombinant bacteria had greater antifungal activity than the wild‐type strain. Specifically, the recombinant strain produced approx. 600% more aureofuscin, as quantified by high‐performance liquid chromatography analysis. Significance and Impact of the Study: To our knowledge, this approach has not been attempted in S. aureofuscus before and few genes in the aureofuscin pathway have been cloned and characterized.