Week 1 HW: Final Project Ideas

Improving extremophile enzyme engineering and production with high-throughput screening

(1) What would you like to biomanufacture and why?

Climate change and industrial contamination are global threatS. Some of the causes lie in the way we are manufacturing. Many industrial processes use chemical catalysts and handle many toxic compounds and solvents, resulting in problematic wastewaters and a potential risk for employees. Additionally, many reactions are carried out under high conditions of temperature and pressure, increasing the energy costs to keep the right environment. The increasing use of enzymes as biocatalysts present benefists to the environment, protection to the working staff and reduction of costs, as the conditions to generate the conversion of reactives requires less dangerous solvents, lower values of temperature and pressure and have a better performance for enantiomer specificity.

Most of the commercial enzymes come from mesophyles, obtaining biocatalysts that performs well between 20ºC and 45ºC. However, they usually underperform with industrial settings. While researchers could engineer these enzymes to give them desired properties. A different approach is the use of extremophile enzymes, with best conditions between 50ºC and 80ºC, often tolerant to denaturing solvents and high pressure. Increasing temperature in industrial processes also increases the solubility of substrate and products. While these natural enzymes are attractive, many features could be enhanced with protein engineering techniques so they can keep their activity under more difficult conditions or improve their specificity for the substrate, etc. Compared with complex gene editing operations, the generation of a large number of random mutations can greatly reduce the difficulty of cell renovation and DBTL cycles, but the accompanied low positive mutation rate needs high-throughput screening (HTS) technology, through which, the researchers can identify the expected mutants with ideal properties. Simultaneously, adopting iterative DBTL rounds, the goal of customizing enzymes that can meet the demand of various industrial processes can be achieved.

To improve the conditions of manufacturing of intermediates, I am proposing a bench-to-industrial scale approach to engineer extremophile enzymes enhanced by HTS technology applied in different parts of the process and a recombinant expression system with downstream conditions. As conventional chemical synthesis of chiral amines relies heavily on expensive, toxic transition-metal catalysts operating under high pressures of hydrogen gas. I will employ thermo-halophilic imine reductase (IRED) as a proof of concept for a greener, solvent-tolerant chiral amine manufacturing sourced from extremophilic metagenomes by improving selected enzymatic features.

(2) What living system would you use?

To successfully go from a lab flak or microplate to an industrial bioreactor we must consider all the scales in our process. We will use two expression chassis, a cell-free system (CFS) and a yeast one. To maintain high library coverage the previous assembled constructs will undergo ultra-high-throughput transformation employing microfluidic electroporation or multi-well microtiter transformations. Employing an optimized E. coli strain to generate libraries. Then, instead of multi-day microbial cultivation, the DNA will be encapsulated into water-in-oil picodroplets with CFE machinery and isothermal expression reagents. After selection of high-performance variants, the chassis corresponds to a yeast cell (Komagataella pharii). As K. pharii can efficiently secrete recombinant enzymes into the culture broth using signal peptides, simplifying downstream processing by avoiding cell lysis steps. Furthermore, it achieves high dry cell weights (> 100 g/L). The regulated expression employs methanol as an inducer, while at industrial scales represents a risk for its inflammable properties, the correct manipulation and strict guidelines reduces the probability near to zero. There is two ways to express this kind of proteins in a recombinant way. The expression lead by plasmid insertion, and the chromosome inserted expression. Obtaining a high expression yield of our protein, the batch must be harvested, and the cells lysed, with mechanical or chemical methods, going to downstream operations to obtain the final product.

(3) How would you use automation?

Automated Gene Assembly: Variant libraries designed in silico will be assembled using a liquid handling robot or microfluidic DNA assembly chips executing Golden Gate or Gibson Assembly protocols. So we can generate a high coverage of potential mutants.

Enzyme mutants screening: After the expression with the CFE system the droplets will be sorted using absorbance-activated droplet sorting (AADS), used when IRED activity is coupled to NADPH consumption. The droplets are measured via optical fibers built into microfluidic channels and sorted at rates of up to 1000 droplets per second. This technique will allow us to select the variants with high activity under the conditions defined in the droplets (e.g. diluted solvent and high temperature).

Culture media optimization: the project proposes a parallel microbioreactor screening (PMS) using automated 24-48 channel microbioreactor systems, like Hamilton bioREACTOR 48 DS, performing multi-factor DoE obtaining the best conditions for biomass growth under specific temperature, C/N ratios and pH profiles.

(4) How much would you need to make, and how long would it take?

(a) How much?

To demonstrate commercial viability, the scale selected for this project is the annual supply of a key pharmaceutical precursor: (R)-1-Boc-3-aminopiperidine, a high-value chiral amine intermediate used in the synthesis of oncology and metabolic therapeutics, such as Linagliptin derivatives.

The global market demand for (R)-1-Boc-3-aminopiperidine is estimated at approximately $250 metric tons (MT) per year (250,000 kg/year) according to a market research report across pharmaceutical manufacturing networks. The proposed biocatalytic process aims to capture $10 of global market demand in its initial commercial activities. Obtaining a required target product quantity of 25000 Kg/year.

The molecular weight of (R)-1-Boc-3-aminopiperidine is 200.28 g/mol. The moles required per year are 25000 Kg/year divided by 200280 Kg/mol, obtaining 124 825 245 mol/year. Considering a transformation rate of 50 000 mol product/mol enzyme, we will need 2 496 mol enzyme/year. Using the estimated molecular weight of engineered IRED (estimated with algorithms with the wild FASTA sequence): 35000 g/mol. Each year, our project must produce 87 377 500 g of enzyme.

(b) How long?

Using HTS technology the enzyme engineering time has significantly decreased from years to months, in some cases even weeks. The project expects to accomplish this time for each enzyme using our screening and iterative system. When we select the best variant, the time to manufacture the proposed amount of enzyme would require nearly a year. Unfortunately I lack the knowledge to correctly calculate the parameters of time and industrial yield, but certainly the optimization of media culture will be decreased using a parallel microbioreactor system and multi-factorial DoE.

5. What don’t you know yet?

One technical challenge comes from the nature of the enzyme selected. Imine reductases rely on the reduced nicotinamide cofactor NADPH. At scale, adding stoichiometric amounts of NADPH is cost-prohibitive. Furthermore, NADPH is thermally labile and rapidly degrades at temperatures above 50 ºC. The use of this cofactor requires a regeneration system employing other enzymes like Glucose Dehydrogenase or Formate Dehydrogenase, which must come from extremophile sources to perform well under the proposed conditions or being equally engineered. The scale-up process often reduces protein yields compared to benchtop microbioreactors, for the localized shear stress mixing gradients in high-capacity bioreactors. This requires an intensive analysis of performance under different conditions and add intermediate steps in the scale-up process.

What would you need to learn, measure, or experimentally determine to know whether your idea could actually work?

Experimentally determine: library quality and diversity, enzyme activity and specificity under solvent presence and high temperature conditions, biomass yield, product recovery, product stability.

Bibliography

Atalah, J., Cáceres-Moreno, P., Espina, G., & Blamey, J. M. (2019). Thermophiles and the applications of their enzymes as new biocatalysts. Bioresource Technology, 280, 478–488. https://doi.org/10.1016/j.biortech.2019.02.008

24chemicalresearch. (2026). (R)-3-Boc-Aminopiperidine market research report 2026-2033 (status and outlook). Recovered on September 2026, https://www.24chemicalresearch.com/reports/307579/bocaminopiperidine-market

Takle, M. J., Maurer, D. M., Staehle, P., Dickhaut, J., Holtze, C., Hellgardt, K., & Hii, K. K. M. (2025). Scalable and sustainable synthesis of chiral amines by biocatalysis. Communications Chemistry, 8(1), 403. https://doi.org/10.1038/s42004-025-01783-w

Yan, W., Li, X., Zhao, D., Xie, M., Li, T., Qian, L., Ye, C., Shi, T., Wu, L., & Wang, Y. (2024). Advanced strategies in high-throughput droplet screening for enzyme engineering. Biosensors & Bioelectronics, 248(115972), 115972. https://doi.org/10.1016/j.bios.2023.115972

Ye, J.-W., Lin, Y.-N., Yi, X.-Q., Yu, Z.-X., Liu, X., & Chen, G.-Q. (2023). Synthetic biology of extremophiles: a new wave of biomanufacturing. Trends in Biotechnology, 41(3), 342–357. https://doi.org/10.1016/j.tibtech.2022.11.010

Final Project