Sung Sun Yim

Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum

Corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. To extend its product spectrum and improve productivity, C. glutamicum needs to undergo further engineering, including the development of applicable promoter system. Here, we isolated new promoters from the fully synthetic promoter library consisting of 70‐bp random sequences in C. glutamicum. Using green fluorescent protein (GFP) as a reporter, highly fluorescent cells were screened from the library by fluorescent activated cell sorting (FACS). Twenty potential promoters of various strengths were isolated and characterized through extensive analysis of DNA sequences and mRNA transcripts. Among 20 promoters, 6 promoters which have different strengths were selected and their activities were successfully demonstrated using two model proteins (antibody fragment and endoxylanase). Finally, the strongest promoter (PH36) was employed for the secretory production of endoxylanase in fed‐batch cultivation, achieving production levels of 746 mg/L in culture supernatant. This is the first report of synthetic promoters constructed in C. glutamicum, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of C. glutamicum. Biotechnol. Bioeng. 2013;110: 2959–2969.

Evaluation of intracellular lipid bodies in Chlamydomonas reinhardtii strains by flow cytometry.

A comparative study of Chlamydomonas reinhardtii wild type CC124 and a cell wall-less mutant sta6-1 is described using FACS in conjunction with two different lipophilic fluorescent dyes, Nile Red and BODIPY 505/515. The results indicate that BODIPY 505/515 is more effective for the vital staining of intracellular lipid bodies and single cell sorting than Nile Red. While BODIPY 505/515 stained cells continued to grow after single cell sorting using FACS, Nile Red stained cells failed to recover from sorting. In addition, a comprehensive study was performed to establish a quantitative baseline for future studies for either lipid accumulation and/or microalgal growth by measuring various parameters such as cell count, size, fatty acid contents/composition, and optical/confocal images of the wild type and mutant.

Systematically programmed adaptive evolution reveals potential role of carbon and nitrogen pathways during lipid accumulation in Chlamydomonas reinhardtii.

BackgroundThe concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions.ResultsThe successive sorting and regeneration of the top 25,000 high-lipid content cells of CC124 and sta6-1, combined with nitrogen starvation, led to the generation of a new population with an improved lipid content when compared to the original populations (approximately 175% and 50% lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes, and the enzymes involved in carbon fixation and uptake, including chlorophyll-ab-binding proteins and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically overexpressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution.ConclusionsAdaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning, flux, and fixation and carbon-nitrogen metabolism during lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation.

Development of a secretion system for the production of heterologous proteins in Corynebacterium glutamicum using the Porin B signal peptide

Corynebacterium glutamicum is one of the useful hosts for the secretory production of heterologous proteins because of intrinsic attributes such as the presence of few endogenous proteins and proteases in culture medium. Here, we report the development of a new secretory system for the production of heterologous proteins by using the porin B (PorB) signal peptide in C. glutamicum. We examined two different endoxylanases and an antibody fragment (scFv) as model proteins for secretory production. In the flask cultivations, all the examined proteins were successfully produced as active forms into the culture medium with high efficiency. For the high-level production of endoxylanase, fed-batch cultivation was also performed in a lab-scale (5L) bioreactor, and the endoxylanases were efficiently secreted in the culture medium at levels as high as 615mg/L. From the culture supernatant, the secreted endoxylanases could be purified with high purity via one-step affinity column chromatography.

Modular Optimization of a Hemicellulose-Utilizing Pathway in Corynebacterium glutamicum for Consolidated Bioprocessing of Hemicellulosic Biomass

Hemicellulose, which is the second most abundant polysaccharide in nature after cellulose, has the potential to become a major feedstock for microbial fermentation to produce various biofuels and chemicals. To utilize hemicellulose economically, it is necessary to develop a consolidated bioprocess (CBP), in which all processes from biomass degradation to the production of target products occur in a single bioreactor. Here, we report a modularly engineered Corynebacterium glutamicum strain suitable for CBP using hemicellulosic biomass (xylan) as a feedstock. The hemicellulose-utilizing pathway was divided into three distinct modules, and each module was separately optimized. In the module for xylose utilization, the expression level of the xylose isomerase (xylA) and xylulokinase (xylB) genes was optimized with synthetic promoters of different strengths. Then, the module for xylose transport was engineered with combinatorial sets of synthetic promoters and heterologous transporters to achieve the fastest cell growth rate on xylose (0.372 h(-1)). Next, the module for the enzymatic degradation of xylan to xylose was also engineered with different combinations of promoters and signal peptides to efficiently secrete both endoxylanase and xylosidase into the extracellular medium. Finally, each optimized module was integrated into a single plasmid to construct a highly efficient xylan-utilizing pathway. Subsequently, the direct production of lysine from xylan was successfully demonstrated with the engineered pathway. To the best of our knowledge, this is the first report of the development of a consolidated bioprocessing C. glutamicum strain for hemicellulosic biomass.

Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum

Corynebacterium glutamicum, which has been for long an industrial producer of various l‐amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large‐scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α‐amylase, and camelid antibody fragment (VHH). For large‐scale production, fed‐batch cultivations were also conducted and high yields were successfully achieved—as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α‐amylase), and 1.57 g/L (VHH)—in the extracellular medium. From the culture media, all model proteins could be simply purified by one‐step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum. Biotechnol. Bioeng. 2016;113: 163–172.

Quantified High-Throughput Screening of Escherichia coli Producing Poly(3-hydroxybutyrate) Based on FACS

Here, we report on a highly sensitive method for the detection of P(3HB) accumulation in Escherichia coli cells based on the automated flow cytometry system using fluorescent dyes. E. coli containing P(3HB) were stained with either BODIPY or Nile red fluorescent dye, and their staining properties were analyzed under a variety of conditions. Compared with Nile red, BODIPY was much more sensitive in staining P(3HB) and overall demonstrated a more rapid staining of cells, a greater resistance to photobleaching, and greater cell viability. In addition, we also successfully monitored heterogeneity in P(3HB) accumulation within a cell population using BODIPY staining and flow cytometry. We believe this optimized staining method using BODIPY in combination with screening by high-speed flow cytometer will be helpful in the engineering of host cells toward an enhanced production of bioplastics.

Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements)

BackgroundIn most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain.ResultsFrom the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA).ConclusionsOur findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C. glutamicum, emphasizing the importance of developing IS element free host strains.

Engineering of Corynebacterium glutamicum for Consolidated Conversion of Hemicellulosic Biomass into Xylonic Acid

Xylonic acid is a promising platform chemical with various applications in the fields of food, pharmaceuticals, and agriculture. However, in the current process, xylonic acid is mainly produced by the conversion of xylose, whose preparation requires substantial cost and time. Here, Corynebacterium glutamicum is engineered for the consolidated bioconversion of hemicellulosic biomass (xylan) into xylonic acid in a single cultivation. First, for the efficient conversion of xylose to xylonic acid, xylose dehydrogenase (Xdh) and xylonolactonase (XylC) from Caulobacter crescentus are evaluated together with a previously optimized xylose transporter module (XylE of Escherichia coli), and cells expressing xdh and xylE genes with an optimized expression system can produce xylonic acid from xylose with 100% conversion yield. Next, to directly process xylan as a substrate, an engineered xylan-degrading module is introduced, in which two xylan-degrading enzymes (endoxylanase and xylosidase) are secreted into the culture medium. The engineered C. glutamicum successfully produce 6.23 g L-1 of xylonic acid from 20 g L-1 of xylan. This is the first report on xylonic acid production in C. glutamicum and this robust system will contribute to development of an industrially relevant platform for production of xylonic acid from raw biomass.

Development of a potential stationary-phase specific gene expression system by engineering of SigB-dependent cg3141 promoter in Corynebacterium glutamicum.

Corynebacterium glutamicum is a non-pathogenic, non-sporulating Gram-positive soil bacterium that has been used for the industrial production of various proteins and chemicals. To achieve enhanced and economical production of target molecules, the development of strong auto-inducible promoters is desired, which can be activated without expensive inducers and has significant advantages for industrial-scale use. Here, we developed a stationary-phase gene expression system by engineering a sigma factor B (SigB)-dependent promoter that can be activated during the transition phase between exponential and stationary growth phases in C. glutamicum. First, the inducibilities of three well-known SigB-dependent promoters were examined using super-folder green fluorescent protein as a reporter protein, and we found that promoter of cg3141 (Pcg3141) exhibited the highest inducibility. Next, a synthetic promoter library was constructed by randomizing the flanking and space regions of Pcg3141, and the stationary-phase promoters exhibiting high strengths were isolated via FACS-based high-throughput screening. The isolated synthetic promoter (P4-N14) showed a 3.5-fold inducibility and up to 20-fold higher strength compared to those of the original cg3141 promoter. Finally, the use of the isolated P4-N14 for fed-batch cultivation was verified with the production of glutathione S-transferase as a model protein in a lab-scale (5-L) bioreactor.