Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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14 | DECEMBER 2017 | | Genetic Engineering & Biotechnology News Nature, as represented by microbial organisms, is up for human-engineered improvement—and not just the sort of improvement that can be achieved by mere tinkering. Craft- work in the modification of microbial organisms is fast giv- ing way to industrial-design approaches. Microbial organ- isms are being stripped down and streamlined so they can be equipped with new modules and "tricked out." These approaches are being implemented systematically by synthetic biology startups. A few representative start- ups—Synthetic Genomics, Synlogic, and Zymergen—are highlighted in this article. All of them are converting microbi- al organisms into bioengineering platforms that can support a design-build-test-repeat approach to product development. Each startup, however, is taking this iterative approach in a different direction. Synthetic Genomics is developing protein-expression plat- forms for the biotech industry. Synlogic is producing "living medicines," probiotics for patients with metabolic disorders or autoimmune disease. Zymergen is developing a platform that can generate specialty materials of interest to diverse in- dustries. These startups often task themselves with squeezing more performance out of microbial organisms that have already been optimized. In a sense, the startups are trying to "gild refined gold." The pursuit is by no means futile. By com- bining biology with automation and data-science strategies, the startups are increasing product output and reducing pro- duction costs while scaling processes from the bench to the bioreactor. Cell Lines on the Assembly Line "When we consider what we are doing now to enhance living systems, we have to look back at history," states Mat- thew Weinstock, Ph.D., a scientist at Synthetic Genomics. "In the early 1940s, producing penicillin meant taking a liv- ing system off the rack and coaxing it to produce at a larger scale. Then in the 1970s, scientists developed protocols for recombinant DNA that allowed one, two, or maybe a hand- ful of modifications. Now we consider a bacterium as a chas- sis and perform large-scale engineering on it to make sweep- ing changes." Synthetic Genomics, through its SGI-DNA subsidiary, genetically engineered Vmax TM , a bacterial host organism designed for improved recombinant protein expression. The Vmax chassis is Vibrio natriegens, a bacterium initially isolated from salt marsh mud in Sapelo Island, GA. It was originally classified as Pseudomonas natriegens, but after it was characterized further, it was recognized as a facultative anaerobe and reassigned to the genus Vibrio. V. natriegens fit the bill for Synthetic Genomics because it is nonpathogenic and the fastest growing bacterium known. Some strains have a doubling time of about 10 minutes, a figure indicating that this bacterium can reproduce two to three times faster than Escherichia coli, the traditional go-to bioproduction workhorse (Figure 1). V. natriegens is supe- rior to E. coli in other respects: It grows to a higher density in culture, it produces more biomass, and it uses cheaper car- bon sources to fuel its growth. All of these features, notes Dr. Weinstock, can "help drive down production costs." Vmax is compatible with most common expression plasmids and promoters used in E. coli for protein expression. Under normal lab conditions at 30 °C, Vmax can produce twice the biomass and four times the protein versus E. coli. That temperature also optimizes pro- tein expression and solubility, while slightly slowing growth. In shake flasks, Vmax expresses higher levels of soluble re- combinant proteins than does E. coli. "It's not a picky eater," states Dr. Weinstock, who points out that Vmax can grow on a wide variety of carbon sources, including sucrose. The Synthetic Genomics scientists who engineered Vmax had to grapple with the usual bottlenecks in protein produc- tion: the need to wait for cultures to reach a high enough den- sity to induce, compounded by the need to wait for enough cell growth to obtain sufficient biomass. To obtain greater yields of soluble protein, scientists optimized a strain of V. natriegens by removing selected pathways and inserting new synthetic DNA sequences to replace some endogenous ones: • A highly regulated, IPTG (isopropyl β-D-1- thiogalactopyranoside)-inducible T7 RNA polymerase was introduced (analogous to the T7-expression system found in some E. coli strains), allowing for higher expression of proteins of interest. • Nucleases and proteases were removed to maintain in- tegrity of isolated plasmid DNA and to improve integrity of soluble protein, respectively. ("Since V. natriegens evolved to deal with harsh environmental insults, for our produc- tion purposes we removed pathways that aren't necessary in a controlled laboratory environment in order to maximize the availability of cellular resources for our desired produc- tion goals," states Dr. Weinstock.) • Catalase, an enzyme that reduces hydrogen peroxide, was added to some strains to improve strain robustness for laboratory growth. If the Vmax is used instead of E. coli, less time is spent streaking plates for sufficient colonies, inoculating cultures, and isolating plasmid DNA. Up to a day can be shaved off a production cycle run. Vmax is also compatible with E. coli expression vectors, antibiotics, and growth media. For future challenges in the field of biomanufacturing, Dr. Weinstock sees improving expression of human proteins in bacteria and OMICS Feature To Gild the Microbe Is Anything but Wasteful Figure 1. Vmax, a bacterial host organism designed for recombinant protein expression, outperforms E. coli-based systems, generating soluble proteins faster, more efficiently, and in larger quantities. Developed by SGI-DNA, a subsidiary of Synthetic Genomics, Vmax is derived from the marine microorganism Vibrio natriegens, a fast-growing, nonpathogenic bacterium. Mary Addonizio, Ph.D. At the dawn of the scientific revolution, the idea that humans could improve on nature was deemed foolish, if not blasphemous. It was even said that to paint the lily, or to throw perfume on the violet, or to add another color to the rainbow, would be "wasteful and ridiculous excess." Now we know better.

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