Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering

Pterostilbene is a derivative of resveratrol with a higher bioavailability and biological activity, which shows antioxidant, anti-inflammatory, antitumor, and antiaging activities. Here, directed evolution and host strain engineering were used to improve the production of pterostilbene in Escherichia coli. First, the heterologous biosynthetic pathway enzymes of pterostilbene, including tyrosine ammonia lyase, p-coumarate: CoA ligase, stilbene synthase, and resveratrol O-methyltransferase, were successively directly evolved through error-prone polymerase chain reaction (PCR). Four mutant enzymes with higher activities of in vivo and in vitro were obtained. The directed evolution of the pathway enzymes increased the pterostilbene production by 13.7-fold. Then, a biosensor-guided genome shuffling strategy was used to improve the availability of the precursor L-tyrosine of the host strain E. coli TYR-30 used for the production of pterostilbene. A shuffled E. coli strain with higher L-tyrosine production was obtained. The shuffled strain harboring the evolved pathway produced 80.04 ± 5.58 mg/l pterostilbene, which is about 2.3-fold the highest titer reported in literatures.

Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting E. coli and Neighboring Gram-Positive Pathogens

We demonstrated that we could combine LLB and phage to construct promising novel antimicrobial agents, LLB-phage. The first LLB-phage, lnqQ -T7 phage, can control the growth of both the Gram-negative host strain and neighboring Gram-positive bacteria while preventing the emergence of phage resistance in the host strain.

Complete Genome Sequence of Bacillus cereus Sensu Stricto VKM B-370, Isolated from the Silkworm Bombyx mori

ABSTRACT Bacillus cereus is a Gram-positive rod-shaped spore-forming bacterium widespread in different environmental niches. Here, we report the complete genome sequence of Bacillus cereus VKM B-370 from the All-Russian Collection of Microorganisms, the host strain for bacteriophages vB_BtS_B83, vB_BcM_Sam46, vB_BcM_Sam112, and Izhevsk.

Development of an Inducible Secretory Expression Vector and Host System for High Yield Production of Recombinant Protein

BackgroundEscherichia coli has been the most widely used recombinant protein expression system due to the availability of various protein expression vectors and ease of genetic manipulation. However, recombinant proteins expressed in E. coli are often contaminated with lipopolysaccharide (LPS) highly toxic to humans and must be removed from FDA-approved biologics, a process which requires extensive and expensive procedures. Gram-positive bacteria possess a single layer of cytoplasmic membrane free of LPS which make it ideal for producing recombinant protein. However, a lack of inducible protein expression systems limits a large-scale protein production in Gram-positive bacteria.ResultsThe HptARS is a three-component regulatory system in Staphylococcus aureus which senses extracellular glucose-6-phosphate and activates the uhpT gene promoter to facilitate uptake of extracellular G6P. To construct an inducible and secretory protein expression vector system, the promoter of the uhpT gene and the N-terminal signal peptide sequence of the hlb gene was fused in-frame with a C-terminal 6x-histidine sequence. For constitutive expression, we generated S. aureus expression host strain lacking the uhpT gene which could not uptake extracellular G6P, resulting in constitutive activation of HptARS system. With this newly established expression vector system and host strain, we demonstrated large-scale production of biologically active and highly pure staphylococcal leukotoxin E. ConclusionExtracellular expression of recombinant protein in LPS-free bacteria has a tremendous advantage in industrial production of FDA-approved biologics. Our newly established inducible and secretory expression vector system and S. aureus host strain will be useful to produce recombinant proteins for vaccine applications and other industrial purposes.

Genome Sequence of the Bacteriophage CL31 and Interaction with the Host Strain Corynebacterium glutamicum ATCC 13032

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4%). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage Φ16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the ∆resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

Genome sequencing of the bacteriophage CL31 and interaction with the host strain Corynebacterium glutamicum ATCC 13032

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4 %). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage Φ16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the Δresmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

Harnessing Escherichia coli for bio-based production of formate under pressurized H2 and CO2 gases

ABSRACTEscherichia coli is gram-negative bacterium that is a workhorse of the biotechnology industry. The organism has a flexible metabolism and can perform a mixed-acid fermentation under anaerobic conditions. Under these conditions E. coli synthesises a formate hydrogenlyase isoenzyme (FHL-1) that can generate molecular hydrogen and carbon dioxide from formic acid. The reverse reaction is hydrogen-dependent carbon dioxide reduction (HDCR), which has exciting possibilities in bio-based carbon capture and storage if it can be harnessed. In this study, an E. coli host strain was optimised for the production of formate from H2 and CO2 during bacterial growth in a pressurised batch bioreactor. A host strain was engineered that constitutively produced the FHL-1 enzyme and incorporation of tungsten in to the enzyme, in place of molybdenum, helped poise the reaction in the HDCR direction. The engineered E. coli strain showed an ability to grow under fermentative conditions while simultaneously producing formate from gaseous H2 and CO2 supplied in the bioreactor. However, while a sustained pressure of 10 bar N2 had no adverse effect on cell growth, when the culture was placed at or above 4 bar pressure of a H2:CO2 mixture then a clear growth deficiency was observed. Taken together, this work demonstrates that growing cells can be harnessed to hydrogenate carbon dioxide and provides fresh evidence that the FHL-1 enzyme may be intimately linked with bacterial energy metabolism.