Fuelling the future of sustainable sugar fermentation across generations.

Biomanufacturing in the form of industrial sugar fermentation is moving beyond pharmaceuticals and biofuels into chemicals, materials, and food ingredients. As the production scale of these increasingly consumer-facing applications expands over the next decades, considerations regarding the environmental impact of the renewable biomass feedstocks used to extract fermentable sugars will become more important. Sugars derived from first-generation biomass in the form of, for example, corn and sugarcane are easily accessible and support high-yield fermentation processes, but are associated with the environmental impacts of industrial agriculture, land use, and competition with other applications in food and feed. Fermentable sugars can also be extracted from second- and third-generation feedstocks in the form of lignocellulose and macroalgae, respectively, potentially overcoming some of these concerns. Doing so, however, comes with various challenges, including the need for more extensive pretreatment processes and the fermentation of mixed and unconventional sugars. In this review, we provide a broad overview of these three generations of biomass feedstocks, outlining their challenges and prospects for fuelling the industrial fermentation industry throughout the 21st century.

Practical considerations for delivering on the sustainability promise of fermentation-based biomanufacturing.

Increasingly, bio-based products made via sugar-powered microbial cell factories and industrial fermentation are reaching the market and presenting themselves as sustainable alternatives to fossil and animal-based products. The sustainability potential of biotechnology, however, has been shown to come with trade-offs and cannot be taken for granted. Shared environmental impact hotspots have been identified across industrial fermentation-based products, including biomass production, energy consumption, and end-of-life fate. Based on both these patterns and our direct experience in preparing for the commercial-scale production of Brewed Protein™, we outline practical considerations for improving the sustainability performance of bio-based products made via industrial fermentation.

Alignment of microbial fitness with engineered product formation: obligatory coupling between acetate production and photoautotrophic growth.

BACKGROUND: Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. However, the sustained productivity of genetically engineered strains is often a challenge, as spontaneous non-producing mutants tend to grow faster and take over the population. Novel strategies to prevent this issue of strain instability are urgently needed. RESULTS: In this study, we propose a novel strategy applicable to all microbial production systems for which a genome-scale metabolic model is available that aligns the production of native metabolites to the formation of biomass. Based on well-established constraint-based analysis techniques such as OptKnock and FVA, we developed an in silico pipeline-FRUITS-that specifically 'Finds Reactions Usable in Tapping Side-products'. It analyses a metabolic network to identify compounds produced in anabolism that are suitable to be coupled to growth by deletion of their re-utilization pathway(s), and computes their respective biomass and product formation rates. When applied to Synechocystis sp. PCC6803, a model cyanobacterium explored for sustainable bioproduction, a total of nine target metabolites were identified. We tested our approach for one of these compounds, acetate, which is used in a wide range of industrial applications. The model-guided engineered strain shows an obligatory coupling between acetate production and photoautotrophic growth as predicted. Furthermore, the stability of acetate productivity in this strain was confirmed by performing prolonged turbidostat cultivations. CONCLUSIONS: This work demonstrates a novel approach to stabilize the production of target compounds in cyanobacteria that culminated in the first report of a photoautotrophic growth-coupled cell factory. The method developed is generic and can easily be extended to any other modeled microbial production system.

Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA.

The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10-25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.