Rapid prototyping of microbial cell factories via genome-scale engineering

Abstract

Advances in reading, writing and editing genetic materials have greatly expanded our ability to reprogram biological systems at the resolution of a single nucleotide and on the scale of a whole genome. Such capacity has greatly accelerated the cycles of design, build and test to engineer microbes for efficient synthesis of fuels, chemicals and drugs. In this review, we summarize the emerging technologies that have been applied, or are potentially useful for genome-scale engineering in microbial systems. We will focus on the development of high-throughput methodologies, which may accelerate the prototyping of microbial cell factories.

Tolerance engineering in bacteria for the production of advanced biofuels and chemicals

“…The tremendous potential of global regulators in obtaining complex phenotypes is best executed using the global transcriptional metabolic engineering (gTME) strategy, which involves generating a randomly mutagenized library of a global stress regulator that is then screened for a desired phenotype. Developed in Saccharomyces cerevisiae and E. coli, this method has seen broad use in obtaining tolerance traits [54,55]. Similarly, manipulation of global signaling compounds can also be used in conjunction with improved solvent-tolerance phenotypes, as was done for isobutanol tolerance using cyclic-di-GMP receptors [51].…”

“…Similarly, manipulation of global signaling compounds can also be used in conjunction with improved solvent-tolerance phenotypes, as was done for isobutanol tolerance using cyclic-di-GMP receptors [51]. New technologies for genome editing (e.g., large clustered regularly interspaced palindromic repeats -interference (CRISPRi) libraries [55][56][57]) and targeted gene regulation [58] allow us to target multiple genes simultaneously and provide powerful new approaches in solvent-tolerance engineering. Genome shuffling across multiple strains with desirable phenotypes, coupled with strong selections or screens, can also be a potential approach to obtain robust strains [59].…”

“…Yet, other organisms, such as cyanobacteria, offer autotrophic production of bulk compounds [99,100] and are also being engineered for improved-tolerance phenotypes [21,27,52,53]. With the recent advances in high-throughput genome editing and evolution [55], these non-model microbes can now be seen as attractive new host platforms. Finally, important studies that are yet to be reported are those that will examine the resilience of these phenotypes during scale up into larger production and the impact of improved tolerance on improving the economic value of the process.…”

The emergence of commodity-scale genetic manipulation

“…For example, given a large enough dataset, recently described machine learning meta-algorithms, such as bagging (bootstrap aggregation -a method used to decrease variance of model predictions by averaging misclassification errors) and boosting (used to decrease bias and variance by weighting classifiers based on accuracy) can calculate models with arbitrarily low prediction error by starting from many (thousands of) models that perform only slightly better than random guessing [2]. Similarly, techniques that allow DNA sequence manipulation have revolutionized biology, allowing thousands of DNA oligomers to be designed, synthesized, and tested with very rapid turnaround times and at increasingly lower cost [3,4]. The ability to construct and reprogram genomes has captured the imagination, with comparisons being drawn between the current state of 'synthetic biology' and the formative days of computer science.…”

White biotechnology: State of the art strategies for the development of biocatalysts for biorefining

“…These promising techniques can use alternative systems: i) zinc finger nucleases (ZFNs) which are artificial proteins that combine the DNA binding domain of a zinc finger with a DNA cleavage domain, ii) transcription activator-like effector nucleases (TALENs) containing respectively a DNA binding and cleavage domain and iii) the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPRassociated protein -(CRISPR-Cas) system in which CRISP nuclease is able to cleave a target DNA sequence with a short protospacer adjacent motif sequence (PAM). All systems allow gene interruptions or insertions at a specific locus in a variety of organism(Jakociunas et al , 2015, Oh and van Pijkeren, 2014, Si et al , 2014, Xu et al , 2014.…”