Ribosome Binding Site Sequences Research Articles

Tumor Pre-Targeting System Using Streptavidin-Expressing Bacteria.

A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe. We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed. Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin. We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.

A robust genetic toolbox for fine-tuning gene expression in the CO2-Fixing methanogenic archaeon Methanococcus maripaludis

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼104-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization

BackgroundRaw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value.ResultsIn this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production.ConclusionsThis study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase.

Vibrio natriegens is the fastest-growing bacteria, and its doubling time is less than 10 min. At present, the T7 expression system has been introduced into V. natriegens for heterologous protein expression, including the commercial strain Vmax1 and the variant VnDX,2 which is a backup expression chassis of Escherichia coli BL21(DE3). However, the strength of the existing T7 expression system is not optimal for every recombinant protein. The different expression strengths of T7 RNA polymerase (T7 RNAP) can be obtained by changing the promoter and ribosome binding site (RBS) sequences of T7 RNAP at different transcription and translation levels. In this work, we obtained a robust VnDX variant library with the fine-tuning T7 RNAP using the industrially used enzyme glucose dehydrogenase (GDH) as the reporter protein. Among this library, the variant VnDX-tet, whose promoter of T7 RNAP was changed from PlacUV5 to Ptet, showed that the reporter enzyme GDH activity was increased by 109% by the T7 expression system. Similarly, variants with different T7 RNAP translation levels were obtained by changing RBS sequences upstream of T7 RNAP, and the results showed that the variant VnDX-RBS12/pGDH had the highest GDH activity, which increased by 12.6%. The VnDX variant library constructed in this study with different T7 expression strengths provides a choice for expressing various recombinant proteins, greatly expanding the application of V. natriegens.

One-Pot Biosynthesis of l-Aspartate from Maleic Anhydride via a Thermostable Dual-Enzyme System under High Temperature.

l-Aspartate is an important chemical in the food and pharmaceutical industries. Herein, a dual-enzyme system was constructed to synthesize l-aspartate from maleic anhydride at 50 °C, which can reduce the byproduct production. Maleate transformed from maleic anhydride in the solution was converted into l-aspartate via fumarate catalyzed by maleate isomerase (MaiA) and thermostable aspartase (AspB), respectively. Because MaiA is a rate-limiting enzyme, enzyme activities of various MaiAs were compared, and the efficient and thermostable maleate isomerase AaMaiA from Alicyclobacillus acidoterrestris was chosen. The Kcat/Km value of AaMaiA was 264.4 mM-1 min-1. AaMaiA and AspB were coexpressed in E. coli to produce l-aspartate. To improve the l-aspartate production rate, the ribosome binding site (RBS) sequence located upstream of AaMaiA was optimized and the Tat signal peptide was fused with AaMaiA. The conversion rate was 96% within 60 min, and the intermediate was not detected, the possible reason of which is that high temperature inhibits the activity of bacterial endogenous enzymes, but functional enzymes remain active. Cells from fermentation produced 243.6 g/L (1.83 M) of l-aspartate with a 2 M substrate. Our study revealed an effective method to produce l-aspartate without using gene knockout and provided a strategy for l-aspartate production in the industrial field.

Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food.

Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.

A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis

To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.Graphical

“Nonpolarity paving” in substrate tunnel of a Limnobacter sp. Phenylacetone monooxygenase for efficient single whole-cell synthesis of esomeprazole

Baeyer-Villiger monooxygenase (BVMO) mediated sulfoxidation is a sustainable approach for the synthesis of esomeprazole. In this work, a novel phenylacetone monooxygenase from Limnobacter sp. (LnPAMO) was found to have trace activity for synthesis of enantiopure esomeprazole. Through engineering in the substrate tunnel using a mutagenesis strategy called “nonpolarity paving” and some modifications in cofactor binding domains, a mutant harboring 15 mutations (LnPAMO Mu15) was obtained with 6.6 × 103-fold higher activity to convert omeprazole sulfide into esomeprazole. The activities of the mutant for synthesis of (S)-methyl phenyl sulfoxide and (S)-pantoprazole also increased much, indicating the versatility of the mutant for sulfoxide synthesis. Importantly, no over-oxidation byproduct omeprazole sulfone was detected in the sulfoxidation products by both mass spectrometry and HPLC analysis. Then NADP-dependent Burkholderia stabili formate dehydrogenase was ligated behind Mu15 along with a ribosome binding site sequence in pET-28a for co-expression. By single whole-cell of recombinant Escherichia coli BL21 coexpressing Mu15 and formate dehydrogenase, omeprazole sulfide was efficiently converted into esomeprazole without production of sulfone (16 g/L substrate, enantiomeric excess > 99.9% (S) and > 99% conversion) and the space–time-yield reached 1.67 g product/L/h.

Biosynthesis of Guanidinoacetate by Bacillus subtilis Whole-Cell Catalysis

Guanidinoacetate (GAA) is a naturally occurring amino acid derivative and the direct precursor of creatine, which is widely used in feed additives and the pharmaceutical industry. The current industrial synthesis of GAA is based on chemical methods, which limits the application of GAA. Here, a biological approach is developed for food safety GAA production via whole-cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis. First, we introduced a heterologous arginine: glycine amidinotransferase (AgaT) from Amycolatopsis kentuckyensis into B. subtilis and optimized its expression level using strategies including: promoter optimization, ribosome binding site (RBS) and N-terminal coding sequence (NCS) screening. In order to alleviate the waste of arginine and the inhibition of AgaT by ornithine, we optimized the natural ornithine cycle in B. subtilis. At the same time, the first gene in the glycine degradation pathway was knocked out. After optimization using these strategies, the titer of GAA was 4.26 g/L with a productivity of 0.21 g/L/h in 20 h, which provides a new method for the biosynthesis of GAA.

Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

CRISPR-Based Construction of a BL21 (DE3)-Derived Variant Strain Library to Rapidly Improve Recombinant Protein Production.

Escherichia coli BL21 (DE3) is the most widely used host for recombinant protein expression. However, not every protein can be highly expressed in BL21 (DE3), so individual optimization strategies are often required for different proteins, which is time-consuming and difficult to apply rapidly for industrial production. Constructing more hosts is a good choice to enrich protein expression selection. The expression level of T7 RNAP is the core control node of the pET expression system, so regulating its expression level is an effective way of improving the production of difficult-to-express proteins. Various BL21 (DE3)-derived variant hosts with different translation levels of T7 RNAP could be obtained by changing the ribosomal binding site (RBS) sequences of T7 RNAP in a genome. Here, a BL21 (DE3)-derived variant strain library with different RBS sequences of T7 RNAP was constructed using a base editor and CRISPR-Cas9. Notably, the CRISPR-Cas9 system combined with degenerate primers enabled the construction of an RBS library with 87.5% of the theoretical coverage in single editing, which is more convenient and efficient than the use of a base editor. The expression level of a target gene in the variant strain library ranged from 28 to 220% of the parental strain. Furthermore, a high-throughput host-screening platform for recombinant protein production was constructed, which enabled us to obtain the best expression host for certain target proteins in only 3 days. As a proof of concept, the production of all eight difficult-to-express proteins was greatly improved, including autolytic protein, membrane proteins, antimicrobial peptides, and hardly soluble proteins. Among them, the expression of glucose dehydrogenase in the best host exhibited a 298-fold increase compared to the parental strain. This strategy is simple and effective, requires no advanced equipment, and can be carried out in any laboratory.

OSTIR: open source translation initiation rate prediction.

SummaryTranslation of messenger RNAs into proteins by the ribosome is a fundamental step in gene expression. In bacteria, it is possible to accurately predict the rate of translation initiation from the sequence surrounding a gene’s start codon using thermodynamic models of RNA folding and ribosome binding. These predictions have applications in a range of fields, from systems biology studies that aim to understand and model bacterial physiology to synthetic biology studies that seek to reprogram bacterial cells. For example, metabolic engineers can design ribosome binding site (RBS) sequences to tune the expression of different enzymes in a pathway and thereby optimize the production of a desired chemical compound by cells. OSTIR (Open Source Translation Initiation Rates) is a Python package and command line tool for predicting translation initiation rates in bacteria.

Optimized expression of Hfq protein increases Escherichia coli growth

Escherichia coli is a widely used platform for metabolic engineering due to its fast growth and well-established engineering techniques. However, there has been a demand for faster-growing E. coli for higher production of desired substances. Here, to increase the growth of E. coli cells, we optimized the expression level of Hfq protein, which plays an essential role in stress responses. Six variants of the hfq gene with a different ribosome binding site sequence and thereby a different expression level were constructed. When the Hfq expression level was optimized in DH5α, its growth rate was increased by 12.1% and its cell density was also increased by 4.5%. RNA-seq and network analyses revealed the upregulation of stress response genes and metabolic genes, which increases the tolerance against pH changes. When the same strategy was applied to five other E. coli strains (BL21 (DE3), JM109, TOP10, W3110, and MG1655), all their growth rates were increased by 18–94% but not all their densities were increased (− 12 − + 32%). In conclusion, the Hfq expression optimization can increase cell growth rate and probably their cell densities as well. Since the hfq gene is highly conserved across bacterial species, the same strategy could be applied to other bacterial species to construct faster-growing strains.

Enhancing the Glucose Flux of an Engineered EP-Bifido Pathway for High Poly(Hydroxybutyrate) Yield Production.

BackgroundAs the greenhouse effect becomes more serious and carbon dioxide emissions continue rise, the application prospects of carbon sequestration or carbon-saving pathways increase. Previously, we constructed an EP-bifido pathway in Escherichia coli by combining Embden-Meyerhof-Parnas pathway, pentose phosphate pathway and “bifid shunt” for high acetyl-CoA production. There is much room for improvement in the EP-bifido pathway, including in production of target compounds such as poly(hydroxybutyrate) (PHB).ResultTo optimize the EP-bifido pathway and obtain higher PHB yields, we knocked out the specific phosphoenolpyruvate phosphate transferase system (PTS) component II Cglc, encoded by ptsG. This severely inhibited the growth and sugar consumption of the bacterial cells. Subsequently, we used multiple automated genome engineering (MAGE) to optimize the ribosome binding site (RBS) sequences of galP (galactose: H (+) symporter) and glk (glucokinase gene bank: NC_017262.1), encoding galactose permease and glucokinase, respectively. Growth and glucose uptake were partially restored in the bacteria. Finally, we introduced the glf (UDP-galactopyranose) from Zymomonas mobilis mutase sugar transport vector into the host strain genome.ConclusionAfter optimizing RBS of galP, the resulting strain L-6 obtained a PHB yield of 71.9% (mol/mol) and a 76 wt% PHB content using glucose as the carbon source. Then when glf was integrated into the genome strain L-6, the resulting strain M-6 reached a 5.81 g/L PHB titer and 85.1 wt% PHB content.

Optimization of ribosomal binding site sequences for gene expression and 4-hydroxyisoleucine biosynthesis in recombinant corynebacterium glutamicum

4-Hydroxyisoleucine (4-HIL) has potential value for treating diabetes. α-Ketoglutarate (α-KG)-dependent l-isoleucine dioxygenase (IDO) can convert l-isoleucine (Ile) into 4-HIL. In our previous study, 4-HIL was de novo synthesized from glucose by expressing the ido gene in Corynebacterium glutamicum strain SN01, an Ile producer, and neither Ile nor α-KG was added. In this study, ribosomal binding site (RBS) engineering was applied for gene expression and 4-HIL biosynthesis in C. glutamicum. The 18 tested RBS sequences showed greatly differing strengths for expressing ido, and 8.10–104.22 mM 4-HIL was produced. To supply the cosubstrate α-KG at different levels, the odhI gene was then expressed using the RBS sequences of high, medium, and low strength in the above mentioned optimal strain SF01 carrying R8-ido. However, 4-HIL production decreased to varying amounts, and in some strains, the α-KG was redirected into l-glutamate synthesis. Next, the O2 supply was further enhanced in three ido-odhI coexpressing strains by overexpressing the vgb gene, and 4-HIL production changed dramatically. 4-HIL (up to 119.27 ± 5.03 mM) was produced in the best strain, SF08, suggesting that the synchronic supply of cosubstrates α-KG and O2 is critical for the high-yield production of 4-HIL. Finally, the avtA gene and the ldhA-pyk2 cluster were deleted separately in SF08 to reduce pyruvate-derived byproducts, and 4-HIL production increased to 122.16 ± 5.18 and 139.82 ± 1.56 mM, respectively, indicating that both strains were promising candidates for producing 4-HIL. Therefore, fine-tuning ido expression and the cosubstrates supply through RBS engineering is a useful strategy for improving 4-HIL biosynthesis in C. glutamicum.

Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli

BackgroundAdvantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes.ResultsWe changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173–2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined.ConclusionsOur findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.

Engineering of L-glutamate oxidase as the whole-cell biocatalyst for the improvement of α-ketoglutarate production

L-glutamate oxidase (LGOX) catalyzes the oxidative deamination of l-glutamate to α-ketoglutarate (α-KG) with the formation of ammonia and hydrogen peroxide. Consequently, identifying a novel LGOX with high enzymatic activity is a prime target for industrial biotechnology. In this study, error-prone PCR mutagenesis of Streptomyces mobaraensis LGOX followed by high-throughput screening was performed to yield four single point mutants with improved enzymatic activity, termed F94L, S280T, I282M and H533R. Moreover, site-saturation mutagenesis at these four residues was employed, yielding two additionally improved mutants, termed I282L and H533L. Subsequently, we employed combinatorial mutagenesis of two, three and four point mutants, and the best mutant S280TH533L showed 90 % higher enzymatic activity than the wild-type control. The data also showed that the presence of these point mutations greatly enhanced enzymatic activity, but did not alter its optimum temperature and pH. Furthermore, the S280TH533L mutant had the maximal velocity (Vmax) of 231.3 μmol/mg/min and the Michaelis-Menten constant (KM) of 2.7 mM, which were the highest Vmax and lowest KM values of LGOX reported so far. Finally, we developed a whole-cell biocatalyst for α-KG production by co-expression of both S280TH533L mutant and KatE catalase. Randomized ribosome binding site (RBS) sequences were introduced to generate vectors with varying expression levels of S280TH533L and KatE, and two optimized co-expression strains were obtained after screening. The α-KG production reached a maximum titer of 181.9 g/L after 12 h conversation using the optimized whole-cell biocatalyst, with a molar conversion rate of substrate higher than 86.3 % in the absence of exogenous catalase, while the molar conversion rate of substrate using the wild-type biocatalyst was less than 30 %. Taken together, these data suggest that the engineering of LGOX has great potentials to enhance the industrial production of α-KG.

High-yield and plasmid-free biocatalytic production of 5-methylpyrazine-2-carboxylic acid by combinatorial genetic elements engineering and genome engineering of Escherichia coli

5-Methylpyrazine-2-carboxylic acid (MPCA) is an important pharmaceutical intermediate and is used in the production of hypoglycemic agents and lipid-lowering drugs. This work aimed to develop a whole-cell biocatalytic process for the efficient synthesis of MPCA from 2, 5-dimethylpyrazine (DMP). Firstly, a whole-cell biocatalyst Escherichia coli strain was constructed by plasmid-based expression of xylene monooxygenase (XMO), benzyl alcohol dehydrogenase (BADH), and benzaldehyde dehydrogenase (BZDH) from Pseudomonas putida ATCC 33015, resulting in MPCA titer of 5.0 g/L. Then, the reaction conditions were optimized and the MPCA titer was further increased to 9.1 g/L. Next, the Ribosome Binding Site (RBS) Calculator v2.0 was used to predict and compare the translation initiation rates of the RBS sequences preceding xylM and xylA genes, encoding the two subunits of XMO. By optimizing the RBS sequences preceding xylA, the MPCA titer was increased to 10.2 g/L and the yield of MPCA on DMP reached 0.665 mol/mol. Finally, to achieve plasmid-free production of MPCA, we integrated the genes encoding for XMO, BADH and BZDH in the genome by using CRISPR/Cas9 and further fine-tuned the copy number ratios of xylM and xylA in the genome, improving the MPCA titer to 15.6 g/L and the yield of MPCA on DMP to 1.0 mol/mol. This work developed a high-yield and plasmid-free biocatalysis process for the environmentally friendly production of MPCA with 100% substrate conversion, and paved the way for the commercial production of MPCA in the future.

Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli.

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803.

Synthetic metabolism allows new metabolic capabilities to be introduced into strains for biotechnology applications. Such engineered metabolic pathways are unlikely to function optimally as initially designed and native metabolism may not efficiently support the introduced pathway without further intervention. To develop our understanding of optimal metabolic engineering strategies, a two-enzyme ethanol pathway consisting of pyruvate decarboxylase and acetaldehyde reductase was introduced into Synechocystis sp. PCC 6803. We characteriseda new set of ribosome binding site sequences in Synechocystis sp. PCC 6803 providing a range of translation strengths for different genes under test. The effect of ribosome-bindingsite sequence, operon design and modifications to native metabolism on pathway flux was analysed by HPLC. The accumulation of all introduced proteins was also quantified using selected reaction monitoring mass spectrometry. Pathway productivity was more strongly dependent on the accumulation of pyruvate decarboxylase than acetaldehyde reductase. In fact, abolishment of reductase over-expression resulted in the greatest ethanol productivity, most likely because strains harbouringsingle-gene constructs accumulated more pyruvate decarboxylase than strains carrying any of the multi-gene constructs. Overall, several lessons were learned. Firstly, the expression level of the first gene in anyoperon influenced the expression level of subsequent genes, demonstrating that translational coupling can also occur in cyanobacteria. Longer operons resulted in lower protein abundance for proximally-encoded cistrons. And, implementation of metabolic engineering strategies that have previously been shown to enhance the growth or yield of pyruvate dependent products, through co-expression with pyruvate kinase and/or fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, indicated that other factors had greater control over growth and metabolic flux under the tested conditions.