An autoinducible trp-T7 expression system for production of proteins and biochemicals in Escherichia coli.

Inducible expression systems can be applied to control the expression of proteins or biochemical pathways in cell factories. However, several of the established systems require the addition of expensive inducers, making them unfeasible for large-scale production. Here, we establish a genome integrated trp-T7 expression system where tryptophan can be used to control the induction of a gene or a metabolic pathway. We show that the initiation of gene expression from low- and high-copy vectors can be tuned by varying the initial concentration of tryptophan or yeast extract, and that expression is tightly regulated and homogenous when compared with the commonly used lac-T7 system. Finally, we apply the trp-T7 expression system for the production of l-serine, where we reach titers of 26 g/L in fed-batch fermentation.

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli.

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.

Generation of an E. coli platform strain for improved sucrose utilization using adaptive laboratory evolution.

BACKGROUND: Sucrose is an attractive industrial carbon source due to its abundance and the fact that it can be cheaply generated from sources such as sugarcane. However, only a few characterized Escherichia coli strains are able to metabolize sucrose, and those that can are typically slow growing or pathogenic strains. METHODS: To generate a platform strain capable of efficiently utilizing sucrose with a high growth rate, adaptive laboratory evolution (ALE) was utilized to evolve engineered E. coli K-12 MG1655 strains containing the sucrose utilizing csc genes (cscB, cscK, cscA) alongside the native sucrose consuming E. coli W. RESULTS: Evolved K-12 clones displayed an increase in growth and sucrose uptake rates of 1.72- and 1.40-fold on sugarcane juice as compared to the original engineered strains, respectively, while E. coli W clones showed a 1.4-fold increase in sucrose uptake rate without a significant increase in growth rate. Whole genome sequencing of evolved clones and populations revealed that two genetic regions were frequently mutated in the K-12 strains; the global transcription regulatory genes rpoB and rpoC, and the metabolic region related to a pyrimidine biosynthetic deficiency in K-12 attributed to pyrE expression. These two mutated regions have been characterized to confer a similar benefit when glucose is the main carbon source, and reverse engineering revealed the same causal advantages on M9 sucrose. Additionally, the most prevalent mutation found in the evolved E. coli W lineages was the inactivation of the cscR gene, the transcriptional repression of sucrose uptake genes. CONCLUSION: The generated K-12 and W platform strains, and the specific sets of mutations that enable their phenotypes, are available as valuable tools for sucrose-based industrial bioproduction in the facile E. coli chassis.

Functional genomics for the oleaginous yeast Yarrowia lipolytica.

Oleaginous yeasts are valuable systems for biosustainable production of hydrocarbon-based chemicals. Yarrowia lipolytica is one of the best characterized of these yeast with respect to genome annotation and flux analysis of metabolic processes. Nonetheless, progress is hampered by a dearth of genome-wide tools enabling functional genomics. In order to remedy this deficiency, we developed a library of Y. lipolytica insertion mutants via transposon mutagenesis. The Hermes DNA transposon was expressed to achieve saturation mutagenesis of the genome. Over 534,000 independent insertions were identified by next-generation sequencing. Poisson analysis of insertion density classified ~ 22% of genes as essential. As expected, most essential genes have homologs in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the majority of those are also essential. As an obligate aerobe, Y. lipolytica has significantly more respiration - related genes that are classified as essential than do S. cerevisiae and S. pombe. Contributions of non-essential genes to growth in glucose and glycerol carbon sources were assessed and used to evaluate two recent genome-scale models of Y. lipolytica metabolism. Fluorescence-activated cell sorting identified mutants in which lipid accumulation is increased. Our findings provide insights into biosynthetic pathways, compartmentalization of enzymes, and distinct functions of paralogs. This functional genomic analysis of the oleaginous yeast Y. lipolytica provides an important resource for modeling, bioengineering, and design of synthetic minimalized strains of respiratory yeasts.

Bioengineering triacetic acid lactone production in Yarrowia lipolytica for pogostone synthesis.

Yarrowia lipolytica is an oleaginous yeast that is recognized for its ability to accumulate high levels of lipids, which can serve as precursors to biobased fuels and chemicals. Polyketides, such as triacetic acid lactone (TAL), can also serve as a precursor for diverse commodity chemicals. This study used Y. lipolytica as a host organism for the production of TAL via expression of the 2-pyrone synthase gene from Gerbera hybrida. Induction of lipid biosynthesis by nitrogen-limited growth conditions increased TAL titers. We also manipulated basal levels of TAL production using a DNA cut-and-paste transposon to mobilize and integrate multiple copies of the 2-pyrone synthase gene. Strain modifications and batch fermentation in nitrogen-limited medium yielded TAL titers of 2.6 g/L. Furthermore, we show that minimal medium allows TAL to be readily concentrated at >94% purity and converted at 96% yield to pogostone, a valuable antibiotic. Modifications of this reaction scheme yielded diverse related compounds. Thus, oleaginous organisms have the potential to be flexible microbial biofactories capable of economical synthesis of platform chemicals and the generation of industrially relevant molecules.

Genome-Wide CRISPRi-Based Identification of Targets for Decoupling Growth from Production.

Growth decoupling can be used to optimize microbial production of biobased compounds by inhibiting excess biomass formation and redirect carbon flux from growth to product formation. However, identifying suitable genetic targets through rational design is challenging. Here, we conduct a genome-wide CRISPRi screen to discover growth switches suitable for decoupling growth and production. Using an sgRNA library covering 12 238 loci in the Escherichia coli genome, we screen for targets that inhibit growth while allowing for continued protein production. In total, we identify 1332 sgRNAs that simultaneously decrease growth and maintain or increase accumulation of GFP. The top target sibB/ibsB shows more than 5-fold increase in GFP accumulation and 45% decrease in biomass formation. Overall, our genome-wide CRISPRi screen provides key targets for growth decoupling, and the approach can be applied to improve biobased production in other microorganisms.