Table 1.

Generalizability of selected molecular tools for chemical biotechnology.

Role	Molecular tool	Host cell(s)	Generalizability	References
Expression tuning	Constitutive promoters	S. cerevisiae	Applications in yeast	[4]
Expression tuning and robustness	Codon optimization	Any	Any	[5, 6]
Expression robustness	Insulated bacterial promoters	E. coli	Any bacterial system where a 160 bp promoter is acceptable and elements in the 5’- UTR are not needed	[7]
Expression robustness	Clustered regularly interspaced short palindromic repeat (CRISPR) RNA processing	E. coli, B. subtilis, S. cerevisiae	Any organism in which the Csy4-based processing platform can be functionally expressed	[8]
Expression tuning	Synthetic ribosome binding sites	E. coli	Applications in E. coli	[9]
Expression tuning	RNA control modules based on Rnt1p hairpins	S. cerevisiae	Applications in yeast	[10]
Enzyme scaffolding	RNA-enzyme assemblies	E. coli	Applications in prokaryotes in which enzymes are amenable to tagging with aptamer binding domains	[11]
Enzyme scaffolding	DNA-enzyme assemblies	E. coli	Applications in prokaryotes in which enzymes are amenable to tagging with zinc-finger proteins	[12]
Enzyme scaffolding	Protein scaffold- enzyme assemblies	E. coli, S. cerevisiae	Applications in which enzymes are amenable to tagging with protein scaffold binding domains	[13, 15, 68, 69]
Enzyme scaffolding	Protein microcompart- ments	E. coli	Potential applications in prokaryotes for enzymes that are amenable to tagging with binding domains or localization tags that direct them to protein microcompartments	[70-73]
Enzyme scaffolding	Host-heterologous protein fusions for localization	S. cerevisiae (in this example)	Any application in which a suitable host localization protein can be identified and a functional host protein-heterologous enzyme chimera can be generated	[14]
Enzyme scaffolding	Organelle- targeting tags	S. cerevisiae	Applications in eukaryotes in which an appropriate organelle tag is available and the enzyme(s) is amenable to fusion	[16]
Genetic assembly	One step homologous recombinatio- nbased plasmid assembly or integration	S. cerevisiae	Applications requiring assembly of 8-10 DNA segments in yeast	[17]
Genetic assembly	Iterative homologous recombination- based integration	S. cerevisiae	Applications in yeast in which one-at-a-time integration and selection marker rescue is desired, such as pathway library construction	[18]
Genetic assembly	Combined in vitro and in vivo assembly for genome-scale constructs	S. cerevisiae	Applications requiring assembly of multi kB- MBconstructs in yeast	[19, 74]
Genetic assembly	Construction of synthetic chromosome arms and replacement of endogenous arms	S. cerevisiae	Applications in yeast in which the incorporation of large/many synthetic fragments and/or deletion of many native elements for stability is desired	[20]
Genetic assembly	Recombination of linear fragments	E. coli	Applications in bacteria	[21]
Genome scale diversity generation	Multiplex automated genome engineering	E. coli	Applications in bacteria where genome-scale diversity generation is useful for optimization and a suitable screening method is available to evaluate the resulting large library	[36]
Genome scale diversity generation	Conjugation assembly genome engineering	E. coli	Applications in bacteria where the combination of defined genomic fragments from multiple lineages into a single genome is desired	[37]
Genome scale diversity generation	Continuous recombination	E. coli	Applications in bacteria where continuous recombination is useful for optimization (e.g. by combining multiple lineages) and a suitable screening method is available to evaluate the large library generated	[75]
Genome scale diversity generation	Homologous recombination of synthetic oligonucleotide libraries	S. cerevisiae	Applications in yeast	[40]
Genome scale diversity generation	Inducible doublestranded break and sexual reproduction	S. cerevisiae	Applications in yeast where desired changes can be obtained in a manageable number of manual rounds	[41]
Diversity screening	Yeast 3-hybrid chemical complementation to screen for bond- forming enzymes	S. cerevisiae	Applications in yeast where enzyme tolerates surface display and two substrates between which a covalent bond is formed are amenable to chemical conjugation	[42]
Diversity screening	Small moleculeresponsive synthetic RNA switch	S. cerevisiae	Applications where RNA that binds the molecule is available or can be selected and incorporated into a self-cleaving genetic device	[43]
Genome scale diversity generation and screening	Phage-assisted continuous evolution	E. coli	Applications in bacteria where the activity of the enzyme to be evolved can be linked to gene expression	[44]
Whole cell gene expression measurement	DNA microarray measurement of global transcriptional response to heterologous protein expression	S. cerevisiae	Applications in yeast where host metabolism or stress response is limiting to strain productivity	[48]
Model-guided host optimization	Target gene knockouts and promoter substitutions based on model prediction	S. cerevisiae	Applications in yeast where host metabolism is limiting to strain productivity	[53]
Model-guided host optimization	Target gene knockouts and gene overexpression based on model prediction	E. coli	Applications in E. coli where host metabolism is limiting to strain productivity	[54]
Dynamic control of gene expression	Glucose responsive promoter	S. cerevisiae	Applications in yeast where linking enzyme expression to glucose levels improves productivity	[64]
Dynamic control of gene expression	Rewired quorum sensing regulon	E. coli	Applications in E. coli where cell-density dependent expression of proteins is desired	[65]
Dynamic control of gene expression	Fatty acid responsive transcriptional regulator	E. coli	Applications in bacteria where gene expression tied to cellular free fatty acid pools is desired and cross-talk between the transcriptional regulator and native transcriptional control is not limiting	[67]