The challenge: Rewire E. coli’s central metabolism – which has evolved over millions of years – to equally prefer xylose and glucose.

Evolution is a powerful tool to rewrite genomes and adapt cells to new environments or new feedstocks. Done by hand, this process requires careful and constant monitoring to keep cultures growing free from contamination and biofilms, for weeks or months at a time.  Software and instrumentation including sensors and microfluidics can supercharge this process by automatically monitoring proliferation rates and recalibrating media and culture conditions with minimal human intervention. With Automated Laboratory Evolution (ALE), fully programmable continuous cultivation of microbial cultures can select for strains that are able to grow in conditions that match the desired technological outcome.

A major chemical company sought to dramatically improve sustainability of commodity bioplastic production by using sugarcane pulp–a waste stream–as a feedstock to produce a critical monomer for plastic in E. coli.  

Efficiently converting to this feedstock, however, requires E. coli to consume both xylose and glucose equally.  While E. coli has the metabolic pathways to consume various sugars, this microbe has evolved to prefer glucose. When glucose and other sugars are present in the media, E. coli consumes all readily available glucose before consuming other sugars. As a result, our partner’s fermentation process took a long time to run. They needed to shorten their fermentation times to meet their target price-points. To achieve this, they needed a new approach to developing their E. coli strain so that it would utilize xylose even in the presence of its preferred carbon source, glucose. 

Our Work

We placed our partner’s strains in different media conditions that mimic the nutrient mix that could come from sugarcane waste. The strain populations that grew on these separate conditions were continuously cultivated for a period of months. During this time, the ALE system automatically determined and adjusted parameters that ensured the cultures maintained a consistent rate of proliferation. This allowed cells to naturally accumulate the mutations that offer a selective advantage and eventually take over the population. The ALE system regularly sampled the cultures and tested their fermentation performance to ensure that the selection experiments were yielding strains that could co-consume glucose and xylose and produce significant amounts of the desired monomer.

Outcome

Amazingly, ALE was able to rewire millions of years of evolution within 5 months with no interruptions arising from contamination or biofilm formation. Strains started to demonstrate increased xylose consumption even in the presence of glucose. The best-performing isolate displayed glucose and xylose consumption at comparable rates. In our customer’s hands, the new strain demonstrated a remarkable 390% increase in xylose uptake in the presence of glucose and reduced batch fermentation time by more than threefold, from 23 hours to 7 hours.

Our Capabilities

Unbiased strain development
Unbiased strain development that takes advantage of natural genetic drift within the population to explore mutations across the entire genome

Evolution of microbial strains
Evolution of microbial strains towards alternative feedstocks and improved tolerance of processing conditions

Fully programmable platform
Fully programmable platform algorithmically adjusts feed rates and gas input to maintain continuous culture of microbial strains for months at a time. The instrumentation and software limit human intervention, keeping cultures free from contamination and biofilms.

Periodic sampling and characterization
Periodic sampling and characterization of strain population to ensure evolved strains have desired fermentation properties