Production of <Emphasis Type="SmallCaps">l</Emphasis>-alanine by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l⁻¹ glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g⁻¹ glucose) with a chiral purity greater than 99.5% l-alanine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) synthesis from glucose

l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS⁻ gluc⁺ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q _l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q _l-Tyr in the PTS⁺ and the PTS⁻ gluc⁺ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS⁻ gluc⁺ tyrR ⁻ strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS⁻ gluc⁺ tyrR ⁻ phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.     

Crystal Structure of the Protein <Emphasis Type="SmallCaps">l</Emphasis>-Isoaspartyl Methyltransferase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Among the known covalent damages that can occur spontaneously to proteins, the formation of isoaspartyl linkages through deamidation of asparagines and isomerization of aspartates may be one of the most rapid forms under conditions of physiological pH and temperature. The protein l-isoaspartyl methyltransferase (PIMT) is thought to recognize l-isoaspartyl residues and repair this kind of damaged proteins. Curiously, there is a potential functional difference between bacterial and mammalian PIMTs. Herein, we present the crystal structure of Escherichia coli PIMT (EcPIMT) at a resolution of 1.8 Å. The enzyme we investigated was able to remain bound to its product S-adenosylhomocysteine (SAH) during crystallization. Analysis indicates that the high affinity of EcPIMT for SAH might lead to the lower activity of the enzyme.     

Enhanced cadaverine production from <Emphasis Type="SmallCaps">l</Emphasis>-lysine using recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> co-overexpressing CadA and CadB

The effect of fusing the PelB signal sequence to lysine/cadaverine antiporter (CadB) on the bioconversion of l-lysine to cadaverine was investigated. To construct a whole-cell biocatalyst for cadaverine production, four expression plasmids were constructed for the co-expression of lysine decarboxylase (CadA) and lysine/cadaverine antiporter (CadB) in Escherichia coli. Expressing CadB with the PelB signal sequence increased cadaverine production by 12 %, and the optimal expression plasmid, pETDuet-pelB-CadB-CadA, contained two T7 promoter-controlled genes, CadA and the PelB-CadB fusion protein. Based on pETDuet-pelB-CadB-CadA, a whole-cell system for the bioconversion of l-lysine to cadaverine was constructed, and three strategies for l-lysine feeding were evaluated to eliminate the substrate inhibition problem. A cadaverine titer of 221 g l^?1 with a molar yield of 92 % from lysine was obtained.     

Functional expression of a human GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose transporter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.

Results

The heterologous expression of the recombinant and codon-adapted human GDP-l-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-l-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of ³H-GDP-l-fucose with a V_max of 8 pmol/min mg with a K_m of 4 µM. The functional expression of SLC35C1 in GDP-l-fucose overproducing E. coli led to the export of GDP-l-fucose to the culture supernatant.

Conclusions

The export of GDP-l-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> W3110 for the production of <Emphasis Type="SmallCaps">l</Emphasis>-methionine

In this study, we constructed an l-methionine-producing recombinant strain from wild-type Escherichia coli W3110 by metabolic engineering. To enhance the carbon flux to methionine and derepression met regulon, thrBC, lysA, and metJ were deleted in turn. Methionine biosynthesis obstacles were overcome by overexpression of metA ^Fbr (Fbr, Feedback resistance), metB, and malY under control of promoter pN25. Recombinant strain growth and methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84p. Blocking the threonine pathway by deletion of thrBC or thrC was compared. Deletion of thrC showed faster growth rate and higher methionine production. Finally, metE, metF, and metH were overexpressed to enhance methylation efficiency. Compared with the original strain E. coli W3110, the finally obtained Me05 (pETMA^Fbr-B-Y/pKKmetH) improved methionine production from 0 to 0.65 and 5.62 g/L in a flask and a 15-L fermenter, respectively.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

Evolving the <Emphasis Type="SmallCaps">l</Emphasis>-lysine high-producing strain of <Emphasis Type="Italic">Escherichia coli</Emphasis> using a newly developed high-throughput screening method

This study provided a new method which applied a selected l-lysine-inducible promoter for evolving lysine industrial strains of E. coli. According to the intracellular levels of the enhanced green fluorescent protein (EGFP) whose expression was controlled by the promoter, 186 strains were preliminarily selected using fluorescence-activated cell sorting from a 10-million-mutant library generated from a l-lysine high-producing E. coli strain. By subsequent multiple parameter evaluation of the 186 selected strains according to the concentration and the yield of lysine, the productivity per unit of cell in 96-deep-well blocks, two mutants MU-1 and MU-2 were obtained. They produced 136.51 ± 1.55 and 133.2 9 ± 1.42 g/L of lysine, respectively, in 5-L jars. Compared with the lysine concentration and the yield of the original strain, those of strain MU-1 improved by 21.00 and 9.05 %, respectively, and those of strain MU-2 improved by 18.14 and 10.41 %, respectively. The mutant selection and evaluation system newly established in our study should be useful for continuous improvement of the current E. coli strains in the lysine industry.     

Spectroscopic,theoretical and structural characterization of hydrogensquarates of <Emphasis Type="SmallCaps">l</Emphasis>-threonyl-<Emphasis Type="SmallCaps">l</Emphasis>-serine and <Emphasis Type="SmallCaps">l</Emphasis>-serine

Summary. Hydrogensquarates of dipeptide l-threonyl-l-serine (H-Thr-Ser-OH) and l-serine (HSq × Ser) have been synthesized, isolated and spectroscopic characterized by solid-state linear-polarized IR-spectroscopy, ¹H- and ¹³C-NMR, ESI-MS and HPLC with tandem masspectrometry (MS-MS) methods. The structures of the salts and neutral dipeptide have been predicted theoretically by ab initio calculations. In the case of H-Thr-Ser-OH the theoretical data are supported by IR-LD ones. The hydrogensquarates consist in positive charged dipeptide or amino acid moiety and negative hydrogensquarate anion (HSq) stabilizing by strong intermolecular hydrogen bonds. The data about the l-serine hydrogensquarate are compared with known crystallographic data thus indicating a good correlation between the theoretical predicted structures and experimentally obtained by single crystal X-ray diffraction.     

<Emphasis Type="SmallCaps">L</Emphasis>-Tyrosine production by deregulated strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.     

Plasmid maintenance and physiology of a genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> strain during continuous <Emphasis Type="SmallCaps">l</Emphasis>-carnitine production

The effect of immobilization on cell physiology and how this determines cell metabolic performance is an important concern for developing bioprocess. This is particularly true for genetically modified microorganisms and their genetic stability. For this reason the stability and physiological state of plasmid-bearing E. coli cells were ascertained by flow cytometry. Differences in the cellular DNA and protein content (15-20%) permit discrimination of control and plasmid-bearing cells, as well as adaptation to continuous cultivation conditions in both freely suspended and immobilized states to be monitored. Moreover, the observed metabolic burden due to maintenance and over-expression of plasmid-coded genetic material and slow cell growth in poorly-viable immobilized cells were found to be the main factors contributing to strain stabilization.     

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose production in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Guanosine 5′-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5′-diphosphate (GDP)-l-fucose. In this study, improvement of GDP-l-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-l-fucose. The effects of overexpression of inosine 5′-monophosphate (IMP) dehydrogenase, guanosine 5′-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine–inosine kinase (Gsk) on GDP-l-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-l-fucose production. Maximum GDP-l-fucose concentration of 305.5 ± 5.3 mg l⁻¹ was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes. Such an enhancement of GDP-l-fucose production could be due to the increase in the intracellular level of GMP.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Enhanced production of GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose by overexpression of NADPH regenerator in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biosynthesis of guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP⁺-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-l-fucose production. However, GDP-l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-l-fucose concentration of 235.2 ± 3.3 mg l⁻¹, corresponding to a 21% enhancement in the GDP-l-fucose production compared with the control strain overexpressing GDP-l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-l-fucose production in recombinant E. coli.     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).