The Inscripta Blog

September 21, 2021

Review of recent achievements in engineering microbial production systems

At Inscripta, we keep our hand on the pulse of recent developments and trends in genome engineering technologies. A recent review article by Shuobo Shi, Nailing Qi, and Jens Nielsen “Microbial production of chemicals driven by CRISPR-Cas systems” published in Current Opinion in Biotechnology caught our attention for focusing on the application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) tools in microbial systems.

The article describes using the Design–Build–Test–Learn (DBTL) cycle for the construction of microbial cell factories. “Traditionally ‘Build’ has been seen as a rate-limiting step in the DBTL cycle,” note the authors. The development of CRISPR tools has changed that. Technologies like the OnyxTM platform that offer automated massively parallel CRISPR editing have revolutionized the throughput and ease of generic manipulation, allowing researchers to focus instead on amassing knowledge of biological systems and developing new applications for microbial cell factories. Below we highlight some notable examples and themes.

Engineering of enzymes for biocatalysis

Enzymes are an important class of bio-based products with many applications from laundry detergents to therapeutics. Ongoing R&D efforts focus on scaling up enzyme production, improving catalytic efficiency or feedback inhibition, and engineering novel functions. Thus, enzyme research includes has both protein engineering and increasing enzyme production.

The authors note that traditional methods for engineering enzymes using plasmid-based systems carry significant disadvantages: “The majority of previous endeavors employed non-integrative plasmids, but suffered from inherent problems of clonal variations, instability, and selection pressure requirements.” CRISPR editing allows to effectively address these issues by enabling interrogation of the protein sequence space in stable genomic expression systems using microbial “work horses”, Escherichia coli and Saccharomyces cerevisiae. The authors use examples from recent publications that demonstrate using CRISPR to improve activity of native and heterologous enzymes.

At Inscripta, we have used Onyx to engineer heterologous protein function and activity in E. coli. In another recent application note, we describe using the Onyx platform to improve production of heterologous cellobiohydrolase I (CBH1) enzyme in S. cerevisiae. CBH1 is actively pursued for its ability to effectively degrade cellulose. In this case, the rate-limiting step is not catalytic efficiency, but expression of this fungal protein in industrially amenable hosts. This involves editing the gene sequence and expression cassette to optimize transcription and translation, as well as introducing genome-wide modifications that improve the overall strain productivity.

Pathway construction and optimization

Like enzymes, small molecule products are also of high commercial interest. These can be common cellular metabolites, natural products obtained by expressing heterologous pathways in recombinant hosts, or synthetic pathways. Genomic integration of non-native pathways has been greatly simplified with CRISPR. Moreover, “optimization of metabolic flux is required for maximizing the [target chemical] production, such as deletion, knock-down or overexpression of target genes, which has been enabled with the assistance of CRISPR technology in a markerless and efficient manner,” note the authors.

Gene expression regulation can be accomplished with tools such as CRISPR interference (CRISPRi) and activation (CRISPRa). The advantage of these approaches is that they could be implemented in multiplex and combinatorial fashion and provide transient regulation that can be used as a metabolic “switch”. For hard-coded regulatory modifications, promoter and ribosomal binding site (RBS) libraries can be used to fine-tune gene expression, which is key to metabolic flux engineering. Our app note on heterologous protein engineering shows an example of high-throughput screening of these regulatory elements which can be used to regulate expression of individual genes and pathways.

Metabolic engineering

The authors also talk about metabolic engineering applications: rewiring cellular metabolism to direct most of the carbon flux to the desired product. Here again, they highlight the need for combinatorial and multiplex editing in order to optimize complex metabolic networks, as well as the advantages of doing this on the genome.

“Because of the complex regulatory metabolic network of living cells, combinatorial and multiplex pathway editing is needed to alter cell metabolism.” Doing this directly on the genome allows to skip the pathway integration steps and shorten the strain development cycles. Additionally, combining rational design with genome-wide exploratory approaches provides an evolutionary advantage and allows to move through the cycle quicker by stacking beneficial edits in iterative rounds of editing.

Read the full review article here.