Multiplex Genome Engineering for Optimizing Bioproduction in Saccharomyces cerevisiae

Abstract

The field of synthetic biology is already beginning to realize its potential, with a wealth of examples showcasing the successful genetic engineering of microorganisms for the production of valuable compounds. The chassis Saccharomyces cerevisiae has been engineered to function as a microfactory for producing many of these economically and medically relevant compounds. However, strain construction and optimization to produce industrially relevant titers necessitate a wealth of underpinning biological knowledge alongside large investments of capital and time. Over the past decade, advances in DNA synthesis and editing tools have enabled multiplex genome engineering of yeast, permitting access to more complex modifications that could not have been easily generated in the past. These genome engineering efforts often result in large populations of strains with genetic diversity that can pose a significant challenge to screen individually via traditional methods such as mass spectrometry. The large number of samples generated would necessitate screening methods capable of analyzing all of the strains generated to maximize the explored genetic space. In this Perspective, we focus on recent innovations in multiplex genome engineering of S. cerevisiae, together with biosensors and high-throughput screening tools, such as droplet microfluidics, and their applications in accelerating chassis optimization.

Improved betulinic acid biosynthesis using synthetic yeast chromosome recombination and semi-automated rapid LC-MS screening

“…Inherent in this approach is bias based on prior knowledge of metabolic networks. An alternative, and complementary, approach is to employ whole-genome mutation strategies and other 'black-box' methods that generate unpredictable alterations to the strain genotype 29,30 . SCRaMbLE is one of the most drastic of these, offering a quick and simple way to explore enormous design spaces of the host genotype 30 .…”

Microbial production of small peptide: pathway engineering and synthetic biology

“…The prepeptide of RiPP is synthetized and modified within the cell, and the mature peptide can subsequently be released by removing the leader peptide through the in vitro cleavage system. (Auxillos et al, 2019). The combination of this technique with suitable screening approaches could be utilized to investigate target strains that possess optimized in vivo metabolic fluxes for target products.…”

“…3 ). Multiplexed automated genome engineering (MAGE) permits access to a library of mutants with diverse genotypes by introducing oligonucleotides, including insertions, deletions and mismatches (Auxillos et al ., 2019 ). The combination of this technique with suitable screening approaches could be utilized to investigate target strains that possess optimized in vivo metabolic fluxes for target products.…”

Whole‐Genome Regulation for Yeast Metabolic Engineering

“…From the experimental data, recombinase activity is entirely limited to designed loxPsym sites, indicating the high fidelity of SCRaMbLE . The diverse SCRaMbLEd strains showed improvement in many complex phenotypes, such as stress resistance (e.g., high temperature, ethanol, and acetic acid) and chemical production …”

“…[87,88] The diverse SCRaMbLEd strains showed improvement in many complex phenotypes, such as stress resistance (e.g., high temperature, ethanol, and acetic acid) and chemical production. [89,90] More recently, researchers have developed several new methods based on SCRaMbLE, including Reporter of SCRaM-bLEd cells using Efficient Selection, Multiplex SCRaMbLE Iterative Cycling, diploid SCRaMbLE, SCRaMbLE-in, in vitro SCRaMbLE, and L-SCRaMbLE, [88,[91][92][93][94][95][96] which explore the SCRaMbLE system in various applications. However, due to the limited number of completed synthetic chromosomes, SCRaMbLE has only been applied in strains with one or two synthetic chromosomes.…”